IL188236A - Phosphoramidate alkylator prodrugs, pharmaceutical compositions containing the same and uses thereof - Google Patents

Abstract

Phosphoramidate alkylator prodrugs can be used to treat cancer when administered alone or in combination with one or more other anti-neoplastic agents.

Description

188236 ρ·Γ» I 453563 τηκ 188236/2 ona ο>ν β¾>™ G»i»3»ii o»ftpii tji»vi.»n ^«ΙΚΛΙΙΙ ono o>>*r>ttnat?ia wv^ ^H Phosphoramidate alkylator prodrugs, pharmaceutical compositions comprising the same and uses thereof Threshold Pharmaceuticals, Inc.

C.1803014 188236/2 BACKGROUND OF THE INVENTION Field of Invention The present invention provides compositions and methods for treating cancer and other hyperproliferative disease conditions with phosphoramidate alkylator prodrugs. The present invention generally relates to the fields of chemistry, biology, molecular biology, pharmacology, and medicine.

Description of Related Art Alkylating agents ("alkylators" or "mustards") used in cancer chemotherapy encompass a diverse group of chemicals that have the ability to alkylate biologically vital maeromolecules such as DNA under physiological conditions (see Hardman et ah, The Pharmacological Basis of Therapeutics, 2001, 1389-1399, McGraw-Hill, New York, USA). DNA alkylation is postulated to be an important mechanism in the antitumor activity of alkylators. The chemotherapeutic alkylators act as strong electrophiles, for example, through the formation of neighboring-heteroatorn-stabilized onium intermediates such as an aziridine or an aziridinium cation.

Phosphoramidate based alkylators used in cancer therapy, such as Cyclophosphamide and Ifosfamide, are an important subclass of chemotherapeutic alkylators. Cyclophosphamide and Ifosfamide are each activated in the liver and the active alkylator released alkylates nucleophilic moieties such as the DNA within the tumor cehs to act as a chemotherapeutic agent. If the active alkylators are released away from the; tumor, DNA and other nucleophilic moieties such as the phosphate, amino, sulfhydryl, hydroxyl, carboxyl and imidazo groups of biomolecules of healthy noncancerous cells, can get alkylated. Such alkylation of healthy cells can and result in unwanted toxic events in patients (see Hardman et al, supra). 188236/2 Cyclophosphamide m Ifosfamide There remains a need for new phosphoramidate based alkylators that can be used to treat cancer or other hyperproliferative disease conditions, preferably compounds less toxic to normal cells. The present invention meets these needs and provides novel phosphoramidate alkylator prodrugs as well as methods of therapy employing them, as summarized in the following section. 188236/2 BRIEF SUMMARY OF THE INVENTION The present invention concerns compounds which are hypoxia activated phosphoramidate alkylator prodrugs, phaiTnaceutical compositions comprising these compounds, and their use in the manufacture of medicaments for treating cancer.

The present invention provides in accordance with its first aspect a compound of . the formula: wherein each X4 are both CI or Br.

In another aspect, the present invention provides the use of a compound according to the invention in the manufacture of a medicament for cancer treatment In another aspect, the present invention provides a pharmaceutical formulation comprising the compound according to the invention and a pharmaceutically acceptable excipient, carrier, or diluent.

Passages ;of the description which are out of the ambit of the claims do not constitute part of the invention. 188236/2 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates the effect of Compound 25 (50 mg/kg) on tumor growth in the H460 xenograft mouse model.

Figure 2 demonstrates the effect of Compound 25 (100 mg/kg) on tumor growth in the H460 xenograft mouse model.

Figure 3 demonstrates the effect of Compound 25 (150 mg/kg) dosed in combination with CDDP on tumor growth in the H460 xenograft mouse model.

Figure 4 demonstrates the effect of Compound 25 dosed in combination with CDDP on tumor growth in the H460 xenograft mouse model.

Figures 5, 6 and 7 demonstrate the effect of Compound 25 in combination with Gemcitabine on tumor growth in the H460 xenograft mouse model. 188236/2 DETAILED DESCRIPTION OF THE INVENTION The detailed description of the different aspects and embodiments of the present invention is organized as follows: Section I provides useful definitions; Section II describes the compounds of the invention and methods for making them; Section III describes methods of treatment, therapies, administrations, and formulations, employing the compounds of the invention alone or in combination; and Section IV provides examples of synthetic methods and biological assays for the compounds of the invention. This detailed description is organized into sections only for the convenience of the reader, and disclosure found in any section is applicable to any aspect of the invention. 188236/2 I. Definitions The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

[0037] As used herein, "a" or "an" means "at least one" or "one or more."

[0038] "Alkyl" means a linear saturated monovalent hydrocarbon radical or a branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix. As used in this disclosure, the' refixes (Ci-Cqq), Ci-qq , or Ci'-Cqq, wherein qq is an integer from 2-20, have the same meaning. For example, (Ci-C8) alkyl, Ci-8 alkyl, or Cj- C8 alkyl includes methyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, tert-butyl, pentyl, and the like. For each of the definitions herein (e.g. , alkyl, alkenyl, alkoxy, araalkyloxy), when a prefix is not included to indicate the number of main chain carbon atoms in an alkyl portion, the radical or portion thereof will have six or fewer main chain carbon atoms. (Ci-Ce) alkyl can be further optionally be substituted with substituents, including for example, deuterium - ("D"), hydroxyl, amino, mono or di(Ci-C6) alkyl amino, halo, C2-C6 alkenyl ether, cyano, ■ nitro, ethenyl, ethynyl, d'-Cfc alkoxy, Q-Ce alkylthio, -COOH, -CONH2, mono- or di(Ci-C6) alkylcarbox-amido, -SO2NH2, -OS02-(C]-C6) alkyl, mono or di(Ci-C&) alkylsulfonamido, aryl,.heteroaryl, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy or heteroarylsulfonyloxy.

·(. · .. . ' ■ . , V": ' . ■ · . ■ . . . ' I ' ·

[0039] "Alkenyl" means a linear monovalent hydrocarbon radical or a 'branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond, but no more than three double bonds. For example, (02-Οδ) alkenyl includes, ethenyl, propenyl, 1,3-butadienyl and the like. ; Alkenyl can be further optionally be substituted with substituents, including for example, deuterium ("D"), hydroxyl, amino, mono or di(Cj -C6) alkyl amino, halo, C2-C6 alkenyl ether, ,cyano, nitro, ethenyl, ethynyl, C C6 alkoxy, C C6 alkylthio, -COOH, -CONH2, mono- or di(C)-C6) alkyl- carboxamido, -S02NH2, -OS02-(Ci-C6) alkyl, mono or di(Ci-C6) alkylsulfonamido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsulfonyloxy.

[0040] "Alkylator" means a reactive moiety capable of forming a covalent alkyl linkage to macromolecules via an electiOphillic reaction with a nucleophile on the macromolecule. "Phosphoramidate alkylator" means an alkylator for which an aziridine or aziridinium electrophile is present or generated by intramolecular cyclization. 1

[0041] "Alkylene" means a linear saturated divalent hydrocarbon radical having from one to'twelve carbon atoms or a branched saturated divalent hydrocarbon radical having from one to twelve carbon atoms optionally substituted with substituents including for example, deuterium ("D"), hydroxyl, amino, mono or di(Ci-C6)alkyl amino, halo, C2-C6 alkenyl ether, cyano, nitro; ethenyl, ethynyl, Ci-C6 alkoxy, C1-C6 alkylthio, -COOH, -GONH2, mono- or di-(CrC6)alkyl-carboxamido, -S02NH25 -OS02-(CrC6) alkyl, mono or di(Ci-C6) . alkylsulfonarnido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsiilfonyloxy. For example alkylene includes methylene, ethylene, propylene, 2-methyl-propylene, pentylene, hexylene, and the like.

[0042] "Heteroalkylene" has essentially the meaning given above foran alkylene except that one or more heteroatoms (i.e. oxygen, sulfur, nitrogen and/or phosphorous) may be present in the alkylene biradical. For example, heteroalkylene includes, -CH2OCH20-,-CH2CH2OCH2CH2-, -CH2CH2N(CH3)CH2CH2-, -CH2CH2SCH2CH2-. arid the.like.

[0043] "Aryl" refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms which is substituted independently with one to eight substituents, preferably one, two, three, four ot five substituents selected from deuterium ("D"), alkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, acylamino, mono-alkylamino] di-alkylamino, haloalkyl, haioalkoxy, heteroalkyl, COR (where R is hydrogen, alkyl, cycldalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), -(CR'R")n-COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, lcycloalkylalkylj phenyl or phenylalkyl) or -(CR'R")n-CONRxRy (where n is an integer frorri 0 to 5, R' and R" are independently hydrogen or alkyl, and R* and Ry are independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl). hi one embodiment, R and Ry together is cycloalkyl or heterocyclyl. More specifically! the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof.

■I' , '. ·■ ; ' ■ ■' ■ ' ·: . . -. · ■ . : .

[0044] . "Cycloalkyl" refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons. The cycloalkyl group can have one or more double bonds and can also be optionally substituted independently with one, two, three or four substituents selected from " alkyl, optionally substituted phenyl, or -C(0)Rz (where Rz is hydrogen, alkyl, haloalkyl, amino, mono-alkylamino, di-alkylamino, hydroxyl, alkoxy, or optionally substituted phenyl).

Mor e specifically, the term cycloalkyl in eludes, for example, cyclopropyl, cyclohexyl, ·. cyclohexenyl, phenylcyclohexyl, 4-carboxycyclohexyl, 2-carboxamidocyclohexenyl, 2- dimethylaminocarbonyl-cyclohexyl, and the like.

[0045] "Heteroalkyl" means an alkyl radical as. defined herein with one, two or three substituents independently selected from cyano, -ORw, -NR Ry, and -S(0)pRz (where p is an integer from 0 to 2 ), with the understanding that the point of attachment of the heteroalkyl ' radical is through a carbon atom of the heteroalkyl radical. R is hydrogen, alkyl, cycloalkyl, cycloalkyi-alkyl, aryl, aralkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcartyamoyl. R* is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl or araalkyl. Ry is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl. Rz is hydrogen (provided that n is 0), alkyl, cycloalkyl', cycloalkyl-alkyl, aryl, araalkyl, amino, mono- alkylaminOjidi-alkylamino, or hydroxyalkyl. Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and 2-methylsulfonyl-ethyl.. For each of the above, Rw, Rx, Ry, and Rz can be further substituted by amino, halo,.fluoro, alkylamino, di-alkylamino, OH or alkoxy. Additionally, the prefix - indicating the number of carbon atoms (e.g., Ci-Cio) refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -ORw, -NR Ry, or ■■· -S(0)pR2 portions. ■, : ' ' '

[0046] In, one embodiment, R and Ry together is cycloalkyl or heterocyclyl.

[0047] "Heteroaryl" means a monovalent monocyclic, bicyclic or tricyclic radical of 5 to 12 , ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attaclimentipoint of the heteroaryl radical will be on an aromatic ring. The heteroaryl ring is optionally substituted independently with one to eight substituents, preferably one, two, three or four substituents, selected from alkyl, cycloalkyl, cycloalkyl-alkyl, halo, nitro, cyano, ; hydroxyl, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxyj heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl, - (CR'R")n-COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), or - (CR'R")n-CONRxRy; (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, arid Rx and Ry are, independently of each other, hydrogen, alkyl, cycloalkyl,!, cycloalkyl-alkyl, phenyl or phenylalkyl). In one embodiment, Rx and Ry together I i is cycloalkyl or heterocyclyl. More specifically the term heteroaryl includes, but is not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrfolyl, py†azolyl, pyridazinyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyj, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl or benzothienyl, indazolyl, pyrrolopyrymidinyl, mdolizinyl, pyrazolopyridinyl, triazolopyridinyl, pyrazolopyrimidinyl, triazolopyrimidinyl, pyrrolotriazinyl, pyrazolotriazinyl, triazolotriazinyl, pyrazolotetrazinyl, hexaaza-indenly, and heptaaza-indenyl and the derivatives thereof. Unless indicated otherwise, the arrangement of the hetero atoms within the ring can be any arrangement allowed by the bonding characteristics of the constituent ling atoms.

[0048] "Heterocyclyl" or "cycloheteroalkyl" means a saturated or unsaturated non-aromatic cyclic radical of 3 to 8 ring atoms in which one to four ring atoms are heteroatoms selected from O, NR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), P(=O)0Rw, jpr S(0)p (where, p is an integer from 0 to 2), the remaining ring atoms being C, where one or two C atoms can optionally be replaced by a carbonyl group. The heterocyclyl ring can be optionally substituted independently with one, two, three or four substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, mono-alkylamino, di-allcylamino, haloalkyl,, haloalkoxy, -COR (where R is hydrogen;, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl^, -(CR'R")n-COOR (n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl], or -(CR'R")n-CONR Ry (where n is; an integer from 0 to 5, R' and R" are ii independently hydrogen or alkyl, R" and Ry are, independently of each other, hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl ori henylalkyl). More specifically the term heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3-yl, N-methylpyrrolidin-3-yl,.2-pyrrolidon-l -yl, furyl, quinolyl, thienyl; benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1,1-dioxo-hexahydro-l|A6-thiopyran-4-yl, tetrahydroimidazo [4,5-c] pyridinyl, imidazolinyl, piperazinyl, 'and piperidin-2-only and the derivatives thereof. The prefix indicating the number of carbon atoms (e.g., C3-Cio) refers to the total number of carbon atoms in the ; portion of the cycloheteroalkyl or heterocyclyl group exclusive of the number ofheteroatoms.

[0049] "CrQ Acyl" means -CO-(C C6 alkyl), wherein the term alkyl is as defined above.

[0050] "Cj-C6 Heteroacyl" means -CO-(CrC6 heteroalkyl), wherein the term heteroalkyl is as. defined above. :

[0051] ' "Aroyl" means -CO-aryl, wherein the tenn aiyl is as defined above.

[0052] "Heteroaroyl" means -CO-heteroayl, wherein the term heteroaryl is as defined above; Alkylsulfonyloxy, heteroakylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy, arylsulfonyloxy, and heteroarylsulfonyloxy groups can be leaving groups in phosphoramidate alleviators and can be replaced in a cell by nucleic. acids such as DNA or RNA, and imidazoles, carboxylates, or thiols of proteins, causing alkylation and cell death. The rate of reaction of j arious Rsuisulfonyloxy groups with nucleic acids, proteins or water can be modulated depending on for example the electron withdrawing nature and the steric bulk of the Rsul moiety and can provide phosphoramidate alkylatdrs and prodrugs thereof which are more toxic to tumors in general and hypoxic zones of .tumor in particular over healthy cells.

[0054] "Substituents" mean, along with substituents particularly described in the definition of each of the groups above,' those selected from: deuterieum,' -halogen,' -OR', -NR'R", -SR', -SiR'R "R'?\ -OC(0)R', -C(P)R', -C02R>, -CONR'R", -OC(0)NR'R", -NR"C(0)R\ -NR'- C(0)NR"R"', -NR"C(0)2R'J -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', - S(0)R', -S(0)2R\ -S(O) 2NR'R", -r>TR'S(0)2R", -CN and -N02, -R', -N3, perfluoro(Ci- C4) alkoxy, and perfiuoro(Ci-C4) alkyl, in a number ranging from zero to the total number of open valences on the radical; and where R', R" and R"' are independently selected from hydrogen, Cj.g alkyl, cycloalkyl, C2_8-alkenylj C2_3 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-Cj-4 alkyl, and unsubstituted aiyloxy-Qi-4 alkyl, aryl substituted with 1-3 halogens, unsubstituted Cj-g alkyl, C] -g alkoxy or Cj-g thioalkoxy groups, or unsubstituted aryl-Cj-4 alkyl. giOups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7- membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of from 1-4 carbon atoms. Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T2- C(0)-(CH2)q-U3-, wherein T2 and U3 are independently -NH-, -0-, -Cl¾- or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A- . (CH2)r-B-, wherein A and B are independently -CH2-, -0-, -NH-, -S-, -S(O)-, -S(0)2-, -S(O) 2N '- or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH2)s-X5-(CH2)t-, where s and t are independently integers of • ■· from 0 to 3 and Xs is -0-, -NR'-, -S-, -S(O)-, -S(0)2-, or -S(0)2NR'-. The substituent R' in -NR'- and 1S(0)2NR'- is selected from hydrogen or unsubstituted C] -6 alkyl.

[0055] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisome s and individual isomers (e.g.:, separate enantiomers) are all intended to be encompassed within the scope of the present invention. The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive.isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0056] The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monbhydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic; or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric,;malohic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, inethanesulfonic, and the like. Also included are salts of amino acids such as argiriate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge, S.M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. ,

[0057] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating tire parent compound in the conventional manner. The parent form of the'compound differs, from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0058] Certain compounds of the present invention can exist in unsolvated fonns as well as splvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or ! ■' . . . 13 amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0059] As used herein, a "glucose analog" includes mono-, di- and tri-saccharides. The glucose analog includes sacchrides comprising glucosamine, N-acetyl-glucosamine; fructose; mannose and mannose derivatives; glucose and glucose derivatives, including but not limited to 2-deoxyglucose (2-DG), N-acetyl-2-amino-2-deoxyglucose, 3-amino-3-deoxy-glucose, 2-amino-2-deoxy-glucose; and galactose and galactose derivatives including but not limited to D-2-deoxy- -galactose, D-4-amino-4-deoxy-galactpse and D-2-amino-2-deoxy-galactose. Thus, the glucose analog can differ from glucose or a derivative such as DG and glucosamine in that it is an epimer thereof. In addition, the glucose analog can be a fluorinated derivative of any of the foregoing compounds. Moreover, the oxygen in the ring of any of the foregoing compounds can be substituted with an isostere selected from the group consisting of S, sulfone, and the like. For example^ , glucose analog can be 5-thio-D-gIucose or a derivative thereof.

[0060] Ajwavy line " means the point of attachment of one group or moiety to another. For example, both and '/ v indicate that the thio group is the point of attachment to another group or moiety.

[0061] The terms CO, C(O), C(=0), -CO- are used interchangeably herein. The terms C02 and COO are used interchangeably herein.. The terms; S02, S(0)2 are used interchangeably herein. The terms SO and S(=0) are used interchangeably herein. The terms PO and P(=0) · are used interchangeably herein.

[0062] As used herein, a "bioisostere" of a chemical moiety such as molecule, group, or atom means another chemical moiety having similar size and spatial disposition of electron pair or pairs.' Bioisosteres and bioisosterism are well-known tools for predicting the biological activity of compounds, based upon the premise that compounds with similar size, shape,, and electron density can have similar biological activity. Known-bioisosteric replacements include, for example, the interchangeability of -F, -OH, - H2, -CI, and -C¾; the interchangeability of -Br and -i-C3H ; the interchangeability of -I and -t-C^; the interchangeability of -0-, -S-, -NH-, -CH2, and -Se-; the interchangeability of -N=,-CH=, and -P== (in cyclic or noncyclic moieties); the interchangeability of phenyl and pyridyl groups; the r ■ f ' · WO 2007/002931 PCT/US2006/025881 interchangeability of -C=C- and -S- (for example, benzene and thiophene); the interchangeability of an aromatic nitrogen (Rar-N(Rar)-Rar) for an unsaturated carbon (Rar- C(=Rar)-Rar); and the interchangeability of -CO-, -SO-, and -S02-. These examples are not limiting on the range of bioisosteric equivalents and one of skill in the art will be able to 5 identify other bioisosteric replacements known in the art See, for example, Patani et'dl., 1996, Chem. Rev. 96:3147-76; and Burger, 1991, J. Prog. Ding Res. 37:287-371.

[0063] A reasonable quantitative prediction of the binding ability or the function of a known molecule can be made based on the spatial arrangement of a small number of atoms or • functional groups i the molecule. As used herein, such an arrangement is called a 0 "pharmacophore", and once the pharmacophore or pharmacophores in a molecule have been identified, this information can be used to identify other molecules containing the same or similar pharmacophores. Such methods are well known to persons of ordinary skill in the art of medicinal chemistry, and as the structural information described in this application identifies the pharmacophore of phosphoramidate alkylator prodrugs and phosphoramidate 15 alkylators . An example of programs available to perform pharmacophore -related searches is the program 3D Pharmacophore search from the Chemical Computing Group (see http://www.chemcomp.com/ fdepfprodinfo.htm).

[0064] ' "Optional" or "optionally" means that the; subsequently described event or circumstance can, but need not, occur, and that the description includes instances where the 20 event or circumstance occurs and instances in which it does not. For example, "heterocyclo group optionally mono- or di- substituted with an alkyl group" means that the alkyl can, but need not be, present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted [with an alkyl group. '■ ' ■ ■ ' .· " '■■' ί ■ ' ■. ·■ ' ■ ■ . : · , , ■ , - 25 [0065] A! combination of substituents or variables is permissible only if such a combination results in aistable or chemically feasible compound. A stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a temperature, of 4 °C or less, in the absence of moisture or other chemically reactive conditions,1 for at least a week.

[0066] As used herein, a "prodrug" means a compound that, after administration, is metabolizek or otherwise converted to an: active: or more active form with respect to at least one biological property, relative to. itself. ' To produce a prodrug, a pharmaceutically active compounder a suitable precursor thereof) is modified chemically such that the modified form is less active or inactive, but the chemical modification is effectively reversible under certain biological conditions such that a pharmaceutically active form of the compound is generated by metabolic or other biological processes. A prodrug can have, relative to the , drug, altered metabolic stability or transport characteristics, fewer side effects or lower . toxicity, o improved flavor, for example {see the reference Nogrady, 1985, Medicinal . Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392). ' Prodrugs can also be prepared using compounds that are not drugs but which upon activation under certain biological conditions generate a pharmaceutically active compound. As used herein- a phosphoramidate alkylator prodrug is a prodrug that upon activation releases the active phosphoramidate alkylator. . [0067] · As used herein, a "cytotoxic agent" is an- agent or compound that produces a toxic effect on cells. As used herein, a "cytostatic agent" is an agent that inhibits or suppresses cellular growth and multiplication.

[0068] As used herein "hypoxic cells" are cells residing in a hypoxic environment in vivo such as, for example, in a hypoxic tumor zone, or in vitro. As used herein "nonnoxic cells" are cells residing in a nonnoxic environment in vivo or in vitro. As used herein "hypoxic cytotoxicity" of a compound or agent is its cytotoxicity on hypoxic cells. As used herein "nonnoxic cytotoxicity" of a compound or agent is its cytotoxicity on normoxic cells.

[0069] As used herein, a "bioreductive group" refers to a group that accepts electrons in an oxidation-reduction reaction. The bioreductive group is a group (1) that can be reduced, i.e., a group that can accept electrons, hydrogen, and/or or an hydride ion; (2) that can be reduced in vivo and/of in vitro; (3) that can be reduced in vivo and/or in vitro under hypoxia; (4) that can be reduced in vivo and/or in vitro by DT-diaphorase, thiols, or by photochemical or electrochemical means; or (5) that can be eliminated and/or cleaved by a biological process, such as by enzymatic hydrolysis, metabolism etc.

[0070] For example, and as described in more detail below, one bioreductive group is a nitroimidazole that may be substituted with a variety of groups. Other examples of bioreductive groups include, but are not limited to, groups based ;on electron deficient nitrobenzenes, electron deficient nitrobenzoic acid amides, nitroazoles, nitroimidazoles, ' 'nitrothiopliehes, nitrothiazoles, nitrooxazoles, nitrofuraris, and nitropyrroles, where each of these classes of moieties may be optionally substituted, such that the redox potential for the bioreductive group lies within a range where the group can undergo reduction in the hypoxic conditions of a rumor, by DT-diaphorase, and/or by a thiol. One of skill in the art will understand, in view of the disclosure herein, how to substitute these and other bioreductive groups to provide a a bioreductive group having a redox potential that lies within said range.

[0071] Generally, one of skill in the art can "tune" the redox potential of a bioreductive group by modifying that group to contain electron withdrawing groups, electron donating groups, or some' combination of such groups. For example, nitrothiophene, nitrofuran, and nitrothiazole groups may be substituted with one or more electron donating groups, including but not limited to methyl, methoxy, or amine groups, to achieve the desired redox potential. In another example, the nitropyrrole moiety can be substituted with an electron withdrawing group, including but not . limited to cyano, carboxamide, -CF3, and sulfonamide groups, to achieve thelidesired' redox potential. For this purpose, strong electron withdrawing groups such as cyano, sulfone, sulfonamide, carboxamide, or -CF3, and milder electron withdrawing groups such as -CKb-halogen where halogen is -F, -CI, or -Br, can be used.

[0072] As used herein, an "anti-neoplastic agent", "anti-tumor agent", or "anti-cancer • agent", refers to any agent used in the treatment of cancer. Such agents can be used alone or in combination with other compounds and can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with neoplasm, tumor or cancer. Anti-neoplastic agents include, but are not'limited to, anti- angiogenic' agents, alkylating agents or alkylators, antimetabolite, certain natural products, platinum coordination complexes, anthracehediones, substituted ureas, rnethylhydrazine derivatives, adrenocortical suppressants, certain hormones and antagonists, anti-cancer polysaccharides, chemoprotectants, and certain herb or other plant extracts.

[0073] As used herein, "cancer" refers to one of a group of more than 100 diseases caused by the uncontrolled growth and spread of abnormal cells that can take the form of solid tumors, lymphomas, and non-solid cancers such as leukemia.

[0074] As used herein, "malignant cancer" refers to cancer cells or cancers that have the capacity of metastasis, with loss of both growth and positional control. 1

[0075] As used herein, "neoplasm" (neoplasia) or "tumor" refers to abnormal new cell or tissue growth, which can be' benign or malignant. ' ' ■ j . ; ( ; . ■ ·

[0076] As used herein, "treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer or other hyperproliferative disease conditions, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, and other beneficial results described below.

[0077] As used herein, "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).

[0078] As used herein, "administering" or "administration of a drug to a subject (and grammatical equivalents of this phrase) includes both direct administration, including self- administration, and indirect administration, including the act of pr escribing a drug. For example, as used herein, a physician who instructs, a patient to self-administer a drug and/or provides a patient' with a prescription for a drug is administering the drug to the patient.'

[0079] As used herein, a "therapeutically effective amount" of a drug is an amount of a drug that, when administered to a subject with cancer, will have the intended therapeutic . . effect, e. g.; alleviation, amelioration, palliation or elimination of one or more manifestations of cancer in the subject. The full therapeutic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.

[0080] As used herein, a "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccun-ence) of disease or symptoms, or reducing the likelihood^of the onset (or reoccurrence) of disease or symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and can occu only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations. • t ; ' .. ·■ · . ' ' · ' .. ' ; " . ., ' '

[0081] As used herein, a "second line" therapy refers to therapy that is given for the treatment of a cancer which has failed to respond to a first chemotherapy regimen or "first line" chemotherapy. "Third line" therapy refers to therapy that is given for the treatment of a - cancer when both initial treatment, first-line therapy, and subsequent treatment, second-line therapy, don't work, or stop working is called.

[0082] As used herein "LogP" means a measure of lipophilicity of a substance determined based on the partitioning of the substance betwen octanol and water.

Ha. Compounds

[0083] Most drug-mediated cancer therapies, including phosphoramidate alkylator based therapies, rely on poisons, called cytotoxic agents, selective for dividing cells targeting, for example, their replicating DNA, microtutbule, and various growth factors and growth factor receptors. These drugs are effective, because cancer cells generally divide more frequently than normal cells. However, such drugs almost inevitably do not kill all of the cancer cells in the patient. ne reason is that cancer cells can mutate and develop drug resistance. Another is that not all cancer cells divide more frequently than normal cells and slowly-dividing cancer cells( can be as, or even more, insensitive to such cytotoxic agents as normal, cells.

[0084] Some cancer cells reside in a poorly vascularized solid tumor, are unable to generate the energy required for cell division and divide slowly. As a tumor grows, it requires a blood supply and,; consequently, growth of new vasculature. The new vasculature that supports rumor growth is often disordered; leaving significant regions of the tumor under-vascularized and even the vascularized regions subjectto intermittent blockage. These. under-vascularized and blocked regions of the tumor become hypoxic - they have a lower oxygen concentration ' · (' .' ■ ■ . · ' . ■ ' ■ ■ ' or a lower oxygen partial pressure.than the corresponding normal tissue, and the cells in them exhibit slower rates of division. Thus, the median oxygen concentration of only ten percent of solid tumors falls; in the normal range of 40 to 60 mm Hg, and fifty percent of solid tumors exhibit median oxygen concentrations of less than 10 mm Hg. t · ·

[0085] The hypoxic areas of the tumor represent a significant source of metastases and cancer cells resistant to therapy (see for example, De Jaeger et ai, Br J Cancer. 2001, 84(9):1280r5 and Rofstad et ai, Br J Cancer. 1999, 80(11):1697-707). Not surprisingly, then, low tumor oxygen levels are associated with a poor response to therapy, increased metastases, and poor survival. The mechanisms of activation and action of Cyclophosphamide and Ifosfamide can exemplify how these agents caninot specifically target the difficult to kill hypoxic zone of a tumor.

[0086] Eloth Cyclophosphamide and Ifosfamide are prodrugs and can be oxidatively activated in the liver via intermediates to yield active phosphoramidate alkylators, Alkylators 1 (cylophosphamide mustard) and 2 (ifosfamide mustard), respectively (see below). The charge neutral Hemiacetals 1 and 2, can have a half life of many minutes and can permeate in and put of the cell. In contrast, the anionic Alkylators 1 and 2 are much less cell membrane permeable and once formed extracellularly inefficiently kills the cell by alkylating cellular DNA. ;

[0087] When the phosphoramidate alkylators reach the tumor, they generally kill cells in the fast growing, well vascularized, normoxic, outer zone of the tumor. However, these phosphoramidate alkylators are not as effective in permeating into the less vascularized, slower growing, progressively hypoxic inner tumor zones and in killing tumor cells therein. Before any of these active alkylators reach the tumor, they can react with healthy cells and Cyclophosphamide Hemiacetal 1 All ylator 1

[0089] The compounds of the invention can generally be described as phosphoramidate alkylator prodrugs. In general, the phosphoramidate alkylator prodrugs of the invention have the following structure Alk-Trigger wherein Alk\is a phosphoramidate alkylator and Trigger T has a structure L-Z3, wherein the linker L is bonded to a bioredtictive group Z3. In one embodiment, the Trigger T is a hypoxia activated trigger.

[0090] Phosphoramidate alkylator derivatives are reported in the references, Borch et al, J. Med. Chem. 2000, 43: 2258-65; 2001, 44: 69-73; 2001, 44: 74-7; Hernick et al. J. Med. ' Chem. 2002, 45: 3540-8; Hernick et al, J. Med. Chem. 2003, 46: 148-54; US Patent Nos.' 4,908,356; 5,306,727; 5,403,932; 5,190,929; 5,472,956; and 6,656,926; US Patent Application Publication No.US.2003/0008850; and Papot et al, Curr. Med. Chem., 2002, 2, 155-85, isolated compounds of which are disclosed therein, and are not the subject of the present invention.. In some embodiments, the phosphoramidate alkylator prodrugs of the present invention have one or more of the following characteristics: (i) a higher hypoxic toxicity or lower value of IC50 or IC90, in hypoxic tissue, (ii) lower normpxic cytoxicity, and (iii) less toxic side effect profile or some combination of these attributes. In some embodiments, the phosphoramidate alkylator prodrugs of the present invention differ from known phosphoramidate alkylator derivatives by: (i) the nature of the phosphoramidate alkylator released, (ii) the nature of the linker (L) and/or the bioreductive group Z3, (iii) the . presence of more than one bioreductive group moiety, or some combination of these .attributes (iv) increased hypoxia selective cytoxicity measured by largerHCR values (v) increased aqueous solubility (vi) increased stability to liver microsomal degradation and/or (vii) providing effective phosphoramide alkylator prodrugs that are achiral and avoid enantiomer specific in vivo metabolism.

[0091] To understand why the prodrug compounds of the present invention represent a significant advance over known anti-cancer phosphoramidate alkylator derivatives, an understanding of tumor biology particularly under hypoxia and phannacokinetics, and phannacodynamics of prodrugs provided herein in particular is helpful.

[0092] For effective tumor therapy, a hypoxia activated prodrug should be much less toxic to healthyjnonnoxic: cells compared to hypoxic tumor cells. In some embodiments, the hypoxia activated prodrugs of the invention are less active and less toxic to normoxic cells than hypoxic cells. When such a prodrug of the invention encounters the hypoxic, reducing , environment within solid tumor tissue, reduction of the bioreductive group causes dissociation of the the phosphoramidate alkylator or the active cytotoxin. The phosphoramidate alkylator is released within the tumor zone and can more easily penetrate the hypoxic iregion of the solid tumor. These phosphoramidate alkylators can kill cells in the difficult to reach hypoxic region of the solid tumor while minimizing death of non-cancerous healthy cells and toxic side effects' to the patient. Thus the present invention provides hypoxia activated prodrugs that are much less toxic to healthy, normoxic cells compared to hypoxic, tumor cells.

[0093] In certain embodiments, the phosphoramidate alkylator prodrugs of the present invention employ nitrp containing aromatic or indole quinone moieries as bioreductive groups in the Trigger T. In the hypoxic tumor, the nitro group is reduced to an hydroxylamino or an amino group and flow of an electron pair from the amino or hydroxylamino group through the conjugated π electron system of the Trigger T releases the phosphoramidate alkylator. In another embodiment, in a hypoxic tumor^ an indole quinone is reduced to an indole hydroquinone and flow of an electron pair from the hydroquinone . through the Trigger T releases the phosphoramidate alkylator. The released phosphoramidate alkylator kills cells in and/or near the hypoxic tumor.

[0094] A number of enzymes can be responsible for the reduction of the bioreductive group r Z3 in the Trigger. For example, cytochrome P450 reductase enzymes can reduce the nitro or a quinone moiety in a bioreductive group in a first- step respectively to a,N (¾('-) or a semi quinone radical anion. The hypoxic tumor zone can have a higher concentration of the reductase enzyme compared to normoxic tissue. Under normoxia, as in well vascularized healthy tissue, in the presence of oxygen; the Q2C-) or the semiquinone radical anion formed can react with oxygen to revert back to the bioreductive group and not ultimately generate or release the phosphoramidate alkylator. The aryl or heteroaryl moiety covalently bonded to the N02(--) or the semiquinone radical anion modulates the oxygen sensitivity of the radical anion.

[0095] The oxygen sensitivity of the bioreductive group varies depending partly on the reduction potential of the bioreductive group. Thus, for example, one bioreductive group can get reduced in a hypoxic tumor zone having ,1 % oxygen, another in a zone having 0.1 % oxygen, and yet another in a zone having 0.01 % oxygen.

[0096] A bioreductive group loses some or all of its hypoxic specificity when it is so easily reduced that the cytochrome P450 reductase enzyme or other reducing agents ("reducing agents") in healthy normoxic tissue can reduce it in the presence of oxygen. If a ΝΘ2('-) or a semiquinone radical anion in a bioreductive group does not react or reacts slowly with oxygen, the radical anion itself can release the phosphoramidate alkylator, or can be further reduced and1 release the phosphoramidate alkylator, causing toxicity to healthy normoxic cells and tissue. The novel phosphoramidate alkylator prodrugs of the presentdnvention are more toxic to the hypoxic cancer cells and tissue compared to the healthy norrnoxic cells and ■ tissue.

[0097] The ease or difficulty of reducing the bioreductive group Z3 can be measured by the reduction potential of the bioreductive group and is influenced by the linker (L), and the phosphoramidate alkylator (Alk-H). For example, attachment of the bioreductive group to an electron withdrawing linker or an electron withdrawing phosphoramidate alkylator can make the bioreductive group easier to reduce compared to when it is covalently bonded to ah electron rich linker or an electron rich phosphoramidate alkylator.

[0098] The Trigger T can be oxidized, hydrolyzed, or thiolyzed and can release the phosphoramidate alkylator in a hypoxia non-senselective manner. Telcyta™, a phosphoramidate alkyltor prodrug that is in the clinic, can release an active toxin in absence of hypoxiafby the action of glutathione transferase (see, e.g., phophoramidate alkylator If in the "Methods of Treatment" section).. The chemical nature of the linker: and/or the phosphoramidate alkylator can influence the oxidative, hydrolytic, or thiolytic stability of the prodrug with respect to phosphoramidate alkylator release. In one embodiment of the present invention a hypoxia activated phosphoramidate alkylator prodrug, does not release the phosphorafnidate alkylator in a hypoxia non-specific, oxidation, hydrolysis, or thiolysis.

[0099] Recording to the present invention, a properly employed Trigger in a phosphoramidate alkylator prodrug can be used to "tune" the pharmacokinetic property of the prodrug without altering its cytotoxic properties. For example, a high volume of distribution of an anticancer agent ensures that the prodrug is absorbed in the tissue guicldy. According to the present invention, in one embodiment, the volume of distribution of a phosphoramidate alkylator prodrug can be modulated by employing a Trigger T containing an amino group : capable of forming an ammonium cation under physiological conditions. In one embodiment, a Trigger T containing a quaternary ammonium group can yield a prodrug ■ compound of the invention having a high volume of distribution while avoiding possible endosomal1 trapping. In another emdodiment, a Trigger T comprising a carboxyl functionality will exist as the anionic carboxylate anion form. C02(")in the extracellular space outside of normal healthy tissue and not pass easily through the normal cell membrane. The lower pH in tumor extracellular space can convert the C02(") to the uncharged "C02H" form allowing the,prodrug to pass through tumor cell membrane.

[0100] A phosphoramidate alkylator containing a hydroxyl, amino, mercapto, and/or a carboxyl group can be transformed into a prodrug by covalently attaching a Trigger T to one or more of these functional groups. During the transformation from a phosphoramidate alkylator to a prodrug, a hydroxyl group in the phosphoramidate alkylator can be transformed, for example, to an ether or an acetal; an amino to an alkylamino, a carbamate, or an amide; a carboxyl group to an ester; and a mercapto group to a thioether or a thioacyl, as described in greater detail in the Method of Synthesis and the Experimental sections below. These transformations can yield a prodrug which is less polar or more lipophilic than the corresponding phosphoramidate alkylator. Non polar phosphoramidate alkylator prodrugs may not be1 readily, soluble in aqueous pharmaceutical carriers or diluents. Solubility /enhancer groups like C02H, amino, allcylamino, dialylainino, and hydroxyl can be employed in the Trigger T to modulate the solubility of the prodrug and overcome any problems encountered in preparing aqueous formulations of the phosphoramidate alkylator prodrugs.

[0101] Phosphoramidate alleviators of the present invention can have one or more N-(2- haloalkyl) or N-(2-haloethyl) a d/or one or more aziridine ("^d) moiety covalently bonded to a P=0 moiety as shown below. Upon release of the anionic phosphoramidate alkylator moiety an aziridine or aziridium species forms which can alkylate DNA (See EXAMPLE section, Example 36). Depending upon the electron withdrawing nature of R2 and R3 substituents, the aziridinium formation kinetics can vary. For example, as shown in the reaction sequence below, the rate of alkylation can increase when the NR2R3 moiety is — -— £, ---- -— - (see Engle ei a/., J.: ¾/. Chem., 1987, 25:1347-57).

Substituents on the nitrogen atoms can alter the geometry of the phosphoramidate alkylator, the derealization of the lone electron pair on this nitrogen atom in the P=0 moiety, the t availability, of the nitrogen lone electron pairs for aziridinium or subsequent aziridine formation, and the aqueous solubility of the phosphoramidate alkylator prodrug and the phosphoramidate alkylator.

[0102] The present invention arises in part out of the discovery that phosphoramidate alkylator prodrugs employing 2-nitroimidazole-bioreductive group show unexpectedly high hypoxic cytotoxicity, low normoxic toxicity and high HCR and improved solubility. For 5, . example, Compounds 24 and 25 were respectively, 400 to 1000 fold more toxic in hypoxic Borch ei a/., J Med:,Chem., andUS Patent No. 6,656,926 both sw -a). 0 alkylators prodrugs wherein

[0104] Y1 is O, S, NR6 or NS02R6 wherein each R6 is independently Ci-C6 alkyl, Ci-C6 heteroalkyl, aryl, or heteroaryl; - wherein eac z, v, q, u, and g independently is 0 or 1 ; , . . ;· ' . 27 29 P36 P37 wherein pJ; is H, Me or allyl;

[0124] 3-(5-Methoxy-l-methyl-4,7-indolequinonyl)-methyl bis[N-methyl-N-(2-bromoethyl)] phosphorodiamidate (P27),

[0125] 3-(5-Methoxy-l -methyl-4,7-indolequihonyl)methyl N,N-bis(2-bromoethyl)-phosphorodiamidate (P2S),

[0126] .. 2-(5-Me1hoxy-l-methyl-4,7-mdo bromoethyl)]phosphorodiamidate (P29), 1

[0127] 2-(5-Meftoxy-l-methyl-4,7-indolequinonyl)methyl N3N-bis(2-chloroethyl) phosphorodiamidate (P30), ; : ,

[0128] 2-(5-Methoxy-l-methyl-4,7-indolequinonyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P31), ' ' \ ■

[0129] 3-(5-Methpxy-l-methyl-4,7-indolequin-onyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P32),

[0130] 2-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl bis[N-methyl-N-(2- bromoethyl)]phosphorodiamidate (P33),

[0132] 2-(5-Memoxy-l -methyl-4,7-indolequinonyl)memyl N,N-bis(2-brom • phosphorodiamidate (P35)

[0133] In a related embodiment, the present invention provides a compound of formula (I) with the proviso that

[0134] (i) at least one of R1-R5 are selected from the group consisting of 2- alkylsulfonyloxyalkyl;, 2-heteroa}kylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl and

[0135] at least one of R1-R5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl ; or

[0136] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Cj- Ce alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-aryisulfonyloxyalkyl, and 2- heteroalkylsulfonyloxj'alkyl; and at least one of NR2R3 and NRiRs is { or

[0137] . (iii) each NR2R3 and NR4R5 are ^J; .

S' ' ' ' ■ ■ .' ' - .

[0138] In another related embodiment, the present invention provides a compound of formula (I) with the proviso that the formula (I) excludes R2 and R3 together forming a morpholine ring or R4 and R5 together forming a morpholine ring.

[0139] Iii one embodiment, the present , invention excludes a compound of the following structure: I „■. -■ . · , · '■■ . · .. ·.' · ' . . wherein Z\ is hydrogen or CrC6 alkyl. [0,140] In; one embodiment, the present invention provides compounds ^wherein the Trigger Tis: [C(Z1)2-Y3]-(C(=0)-0)-[C(Z1)2-Z2-Y4] -[CiZ zl tCCZ KXZ l-Zs; [0.145] . In tone embodiment, -[C(Z1)=C(Z])]- is:-CH=CH-, -C(CN)=CH-, -CH=C(CN)-, - ' . C(Ar)=CI-K -CH=CAr-, -C(COAr)=CH-,-CH=C(COAr)-, -C(COR12)=CH- or - CH=C(CORi2)-, wherein Ar is aryl optionally substituted with up to five substituents selected from the group consisting of OH, OMe, CF3, 0-CHF2, OCF3, N02, CN, halo, halomethyl, 5 dihalomethyl, trihalomethyl, hydroxymethyl, C02H, CONH2, CONMe2, arid CONHMe; and Ri2 is is independently hydrogen, C]-C6 allcyl, Ci-Ceheteroalkyl, C3-C8 cycloalkyl, or heterocyclyl. . . . ■ [0146] I another embodiment, Trigg )2.

[0148] In one embodiment, the present invention provides compounds of formulas (II) and ,;;, · (Hi): ^ .■ ■ (Π) (HI) wherein each R2-Rs independently is selected from the group consisting of hydrogen, hydroxyl, Ci-C6 alkyl, Ci-C6heteroalkyl, C3-C8 cycloalkyl, hetei cyclyl,.Ci-C6 aIkoxy, Ci-C6 alkylamino,i C1-C6 dialkylarninb,, aryl and heteroaryl; or together any two of R2-R5 form a C3- C10 heterocycle; each Y\ independently is S or O; and each Trigger T is defined as in formula (i);

[0149] with ttie proviso that in formulas (II) or (III):

[0150] (i) at least two of R1-R5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2 heteroalkylsulfonyloxyalkyl; of

[0151] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-d- C6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is ; or

[0155] (i) one ofR2 and R3 is H and one ofR4 and R5 is H;

[0156]: (ii) one of R2 and R3 is Q-alkyl and one of R4 and R5 is Cj-alk l; or

[0157] (iii) at least one of R2-R5 is hydroxyl, amino, C3-C8 cycloalkyl, heterocyclyl, Ci-C6 alkoxy, Ci-C6 alkylarnmo, Ci-C6 dialkylamino, aryl, heteroaryl, Ci-C acyl, Ci-C6hetei acyl, or aroyl or heteroaroyl.

[0158] In one embodiment, the present invention provides a compound of formula (II) wherein Z3 is a bioreductive group selected from: with the proviso that in formula (I): ..-

[0159] (i) at least one of Ri -R5 are selected from the group consisting of 2-alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-heteroalkylsulfonyloxyallcyl and

[0160] at least one of R1-R5 are selected from the group consisting of 2-haloalkyl, 2-alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- ·" heteroalkylsulfonyloxyalkyl; or

[0161] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci-Ce alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is ; or

[0162] (Hi) each NR2R3 and MLiRs are

[0163] In one aspect, the present invention provides phosphorainidate alkylator prodrugs of formula (I): ' : (I) wherein -[C(Z])2-Y3]v-(S(=0)2)q-[C(Zi)2-Z2-LY4]u-Z3, wherein each v, q, and u independently is 0 or 1 and Z3 is a glucose or an analog thereof with the proviso that it excludes glucose conjugates of phosphoramidate alkylators described in the reference Wiessler et al., US Patent No. 5,622,936; ( '

[0165] each of R2-R5 independently is hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, C\-C alkoxy, Ci-C6 alkylamino, Ci-C6 dialkylamino, aryl and heteroaryl, C i-C^ acyl, Ci-C&heteroacyl, aroyl, or heteroaroyl; or together any two of R1-R5 form a C3-Ci0 heterocycle;

[0166] with the proviso that in formula (I):

[0167] (i) at least two of R2-R5 are selected from the group consisting of 2-haloalkyI, 2-alkylsulfonyloxyalkyl, 2.-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-heteroalkylsulfonyloxyalkyl;

[0168] (ii) at least one of R2-R5 is selected from the group consisting of 2-haloalkyl, 2-C 1 -C6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is. ; or •[0169] (iii) each NR2R3 and NR4R5 are and "[0170] ah individual isomer or a racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.

[0171] I another embodiment, the present invention provides the compounds: P osphoramidate alylator prodrugs Phosphoramidate alkylator cytotoxins

[0172] Ih another embodiment, the present invention provides the compounds:

[0173] In another embodiment, the present nvent on provides the compoun s: In one embodiment, R6 is -(N-CH2CH2X4)2.

[0174] In another embodiment, the present invention provides the compounds: date alkylator prodrug to yield the corresponding phosphoramidate alkylator. i the compounds: (IV) ■, (V) ' - "5 " !

[0183] In one embodiment, the present invention provides a compound of formula (IV) wherein one of Rio is -(CH2)e-Intercaiator wherein an Intercalator is an aromatic or heteroaromatic moiety capable of intercalating between a nucleic acid base pair.

[0184] In another embodiment, the present invention provides the compound: wherein ¾ and Ri0 is defined as in formula (IV).

[0185] ; In another, embodiment, the present invention provides the compound:

[0186] In one fomiula (VIII): wherein each R9 is independently hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl; and N(Rio)2 is selected from NH2, NHMe, NMe2, NEt2, HOMe, and HOH.

[0187] In one embodiment, the present invention provides the compound of the formula (IX): : (IX) wherein each R9 independently is hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl.

[0188] In one embodiment, the present invention provides the compound of the formula (X): " ' ( ) ■ wherein each R9 independently is hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl; and each Rn is independently hydrogen, methyl, ethyl, propyl, isopropyl, benzyl, substituted methyl, cyclopropyl, methoxy, and hydrpxyl; or together two Rn form a heterocycle.

[0189] In one embodiment, the present invention provides the compounds of the formulas (X-A), (X-B) and (X-C): (X-C) wherein X2 and X4, are defined as in fonnula (I), and R!0, and Rn are defined as in fonnulas (IV), (VI) and (VII).

[0190] In one embodiment, the present invention . provides the compounds of the fonnula (XI) - (XV): ; (XIII) and (XIV) - (XV) . ■ ■ wherein each Ru independently is hydrogen, methyl or substituted methyl, benzyl, isopropyl, propyl, cyclopropyl, methoxy, and hydroxyl; and i, X2, and Z3 are defined as above; and X4 is CI, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, cycloalkylsulfonyloxy, heterocycloalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy. In one embodiment, in compounds of fonnulas (XII), (XIV),· and (XV), when X4 is CI or Br then R] 1 excludes isopropyl.' In one embodiment, a compound of 'formula (X) excludes a compound wherein Z3 ί ' ■ ί WO 200.7/002931 PCT/US2006/025881

[0191] In one embodiment, the present invention provides a compound of formula (XII), (XIV), or (XV) wherein each Rn is hydrogen. Examples of compounds of formula XII, XIV, or XV include compounds 5, 7, .8, 9, 10, 13, 14, 15, 19, 23, 24, 25, 26, 32, 34, and 36. In one embodiment, the present invention provides phosphoramidate alkylator prodrugs of formulas XII, XIV, or XV wherein ¾ j excludes propyl or isopropyl. In another embodiment, the present invention" excludes the compound:

[0192] In one embodiment the present invention provides a phopsphoramidate alkylator prodrug wherein RH is C3-C8 cycloallcyl. In another embodiment, the cycloalkyl is cyclopropyl. In general, a cyclopropyl group can be more stable than ah alkyl group to oxidatively metabolizing proteins in the cell, particularly in the liver the prodrug compounds of the invention provide a pharmacokinetically improved phopsphoramidate alkylator prodrug compared to known phosphoramidate alkylator prodrugs.

[0193] In one embodiment, the present invention provides the compounds of the formula (XVI) , ; wherein K is Ci-Cialkylene or Cj-Ceheteroallcylene. In one embodiment K is (C(Ri2)2)e, CH2CH2(-X6-CH2CH2)f, or CH2(-X6-CH2)f wherein e is 1-10, f is 0-3, and X6 is O, S, or • R12 wherein each R12 is independently defined as above.

[0194] In one embodiment, the present invention provides the compounds of the fonnula (XVII - (XVIII) (XVE) (XVIII) wherein e is 0-4, X4 is CI or Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy; Χβ is O, S, or NR12 wherein R12 is defined as above.

[0195] Iri one embodiment, the present invention provides the compound of fonnula (XIX): (XIX) wherein e is 0-4, and X4 is CI, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy. In a related embodiment, the present invention provides a compound of formula (XIX) wherein e is 1. See EXAMPLE section for examples of compounds of formulas described herein.

[0196] In one embodiment, the present invention provides the compound of formula (XX): (XX) wherein Rg;is glucose or a glucose analog; e is 0-4, and X4 is CI, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroaiylsulfonyloxy. As used herein, a glucose analog includes mono, di and tri saccharides. In a related embodiment, the present invention provides a compound of formula XX wherein e is 1.

[0197] In one embodiment,.the present invention provides the compounds: wherein R9 and 4 are defined as in formula VI.

[0199] In one embodiment, the present invention provides the compound of the formula (XXI) (XXI) .

[0202] In another aspect, the present invention provides the compounds of the formula (xxii): ; wherein

[0203] Ri - Rs, Yi, and Y2 are defined as in formula (I); • [0204] each Ri-R5,and Ri*-Rs* independently is selected from the group consisting of hydrogen, h droxyl, Ci-C6 alkyl, d-C6 alkoxy, Ci-C6 alkylamino, CrC6 dialkylamino, aryl, -[G(ZI)2-Y¾]v-(S(=0)2)q-[G(Z1)2-Z2-Y4]u [C(Z1)2]2-[C(Z,)=C(Z1)]g-Z3-; with the proviso that in formula (XXII): ϊ■

[0205] (i'j at least two of-Ri-R5 and Ri *-¾.* are 2-haloalkyl, 2-all ylsulfonyloxyalkyl, 2. heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2 heteroalkylsulfonyloxyalkyl; or

[0206] (i'i) at least one of Ri-R5 and Ri*-R5* is 2-haloalkyl, 2-CrG6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2-heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR2*R3* is <1: or

[0207] (iii) each NR2R3 and NR2*R3:|: both ; and an individual isomer or a racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.

[0208] each Z independently is C, S, or P;

[0209] each t independently is 1 or 2;

[0210] each r independently is 0 or 1 ; · ' , [0211] K is selected from the group consisting of Ci-C6alkylene, d-Ce heteroalkylene, arylene, or heteroarylene, (C(R9)2)n; and (Y5-(C(R9)2)m- -(C(R9)2)m-Y6)ii wherein n is 1-8;

[0212] each m independently is 1-4;

[0213] each 9 is independently d-Q alkyl or heteroalkyl, or together when covalently bonded to the same carbon atom or adjacent carbon atoms are cycloalkyl or heterocyclyl; and , [0214] , each Y4,Ys, and Y6 independently is O, S, NR7, or a bond; with the proviso that one of Y4,Y5, arid Y6 has to be O, S, or R7.

[0215] In another aspect, the present invention provides the compounds of the formula (XXIII): · ; (XXIII) wherein · , - ; \

[0216] Ri - R5, Yi, and Y2 are defined as in formula (I);

[0217] " each R1-R5 and Ri*-R5* independently is selected from the group consisting of hydrogen, ;hydroxyl, Ci-C6 alkyl, C1-C6 alkoxy, Ci-C6 alkylamino, Cj-Ce dialkylamino, aryl, heteroaryl; or together R2 and R2* form a heterocycle; or each R1-R5* independently is a Trigger T selected from the group consisting of -[C(Z))2-Y3]v-[C(=0)-0]q-[C(Z1)rZ2-Y4]u-[C(Z1)2]z-[-C(Z1)=C(Z!)]g-Z3 and

[0219] (ii) at least one of R2-R5 and R2*-R5* is 2-haloalkyl, 2-C,-C6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2-heteroalkylsulfonyloxyalkyl; and one of NR2R3 and NR2*R3* is <1. or

[0220] (iii) NR2R3 and NR2*R3* together are both or R4R5 and N 4*R5* together are both " 3; and

[0221] ah individual isomer or racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.

[0222] L2 is wherein Xiis defined as above.

[0223] In another embodiment, the present invention provides the compound of the formula (XXIV): ·■ (XXIV) wherein R2, R3, R4, ¾*, R3*, R4*, Z, K and Trigger are as defined in Fonnula (XXII).

[0224] In another, embodiment, th'e present invention provides, the compounds of formula (XXIV) having the structure of formula (XXV) or (XXVI): (XXV) (XXVI)

[0225] In another embodiment, the present invention provides the compound df the formula (XXVI): i wherein Xi, X2, Xt, and e are defined as in formula (XXV).

[0226] Ιτί another aspect, the present invention provides the compound of the formula (XXVII): , R4 R4 f?3 \ / Rs R5 I I ' Trigger^ ^W-^ ^ Trigger .

(XXVII) wherein R2-R5, r, k, Y], and Trigger T are defined as in formula (XXIV).

[0227] lib. one embodiment, the the present invention provides a compound of Formula: WO 2007/002931 ' PCT/US2006/025881 Z3 selected from the group i

[0229] In another embodiment, the present invention provides a moiety having the formula: R4 ¾ I ° I .' 5 P R2 O. \ selected from the group consisting of: wherein each R9 is hydrogen or CrC6 alkyl and each X4 is halo or another embodiment, R9 is hydrogen, methyl, ethyl, isopropyl, or isobutyl; bromo, or methanesulfonyloxy.

[0231] In another embodiment, the present invention provides a compound of formula: wherein In another embodiment, X is CI or Br. Such deuterated phosphoramidate alkylators and their prodrugs are equally cytotoxic with respect to hypoxic tumor tissue as their non-deuterated or hydrogenated analogs, such as compounds 25, 36 and the likes,. However, the presence of such deuterated analogs in vivo, for example in blood plasma, can be determined more efficiently compared to their corresponding » ■ '..·■·- . . ·. · ■ ' , .. . .- phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs by nuclear magnetic * ' · ' ' ■ ■ .■ ■ ■ , resonance methods and such deuterated analogs can be useful in determining pharmacokinetic or pharmacodynamic properties of the phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs. Phannacokinetic and or pharmacodynamic infonnation of phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs is used in determining dosage, frequency of dosing, and similar administration related parameters. The synthesis-of a octadeuterated-compouiid 25 and octadeuterated isofosfamide alkylator is described in the EXAMPLE section. ■j1 ' ' ' 61

[0233] In another group of embodiments, the present invention provides the individual' and selective groupings of the compounds of the EXAMPLES. Examples of compounds of the 64 \ healthy,

[0235] In one embodiment, the present invention provides a novel phosphoramidate alkylator prodrug which upon bioreduction releases the corresponding novel or Icnown phosphoramidate alkylator , NEt2,.: and NHOH; each R11 is independently hydrogen, Me, ethyl, cyclopropyl, isopropyl, propyl, benzyl, substituted methyl, cyclopropyl, methoxy, and hydroxyl; or together two Rn form a heterocycle.

[0237] The anti-cancer agent Cyclophosphamide metabolizes to Id (Rio is hydrogen) and Ifosfamide metabolizes to le (each Rj 1 is hydrogen), when used in cancer treatment.

Glufosfamide, which is being evaluated in the clinic for cancer treatment, releases an alkylator of formula le (eachRn is hydrogen, see Wiessler et al., US Pat. No. 5,622,936; PCT application No. US05/03370-entitled "Anti Cancer Therapies", US Pat. Appl. No. 60/63S995 entitled "Glufosfamide Combination Therapy" and Attorney docket No. 021305-005900US filed on 1 May '2005 entitled "Glufosfamide Combination Therapy"). Telcyta™ which is being evaluated in .the clinic for cancer treatment, releases If (Rosen et al., Clin Cancer Res. 2004, 10(l l):3689-98). .

[0238] Knowii phosphoramidate alkylator prodrugs such as.ifosfamide and cyclophosphamide metabolize to produce cytotoxic by products such as acrolein and chloroacetaldehyde which cause undesirable patient side-effects such as hemorrhagic cystitis, coma or death; In one embodiment, the present invention provides a phosphoramidate " alkylator prodrug which upon metabolism produces less toxic by products per treatment as compared to those produced by the metabolism of ifosfamide and/or cyclophosphamide. In one embodiment, the phosphoramidate alkylator prodrugs of the present invention do not' produce acrolein by in vivo metabolism. Examples of toxic by products resulting from metabolism of the prodrugs of the invention include chloro, bromo, alkylsulfonyloxy, hetero alkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy-acetaldehyde, (for metaboliC'production of chloroacetaldehyde from ifosfamide see the reference Hardman et al, 5iip/'fl,;page.l396). In another embodiment, the present invention provides a phosphoramidate alkylator prodrug which upon oxidative metabolism produces 5-95% as much chloroacetaldehyde or an equivalent as defined above, per treatment, as produced by ifosfamide metabolism.

-[C^-Z^] -[C(¾)2]z-[^^^

[0244] In one embodiment, the present invention provides a Trigger T which upon bioreduction is modified to TriggerMod or T and the phosphoramidate alkylator is separated from T in less than 0; 1 second. In another embodiment, the phosphoramidate alkylator is 10 separated from Tm in between 0.01 to 0.10 second. In another embodiment, the phosphoramidate alkylator is separated from T in between 0.1 to 1,0 second. In another embodiment, the active phosphoramidate is separated from TM in between 1.0 to 10.0 seconds; In another embodiment, the phosphoramidate alkylator is separated from TM in between lOlO to 100.0 seconds.

[0245] In airelated embodiment, upon activation or reduction, a phosphoramidate alkylator prodrug yields a prodrug with a modified Trigger T (TM) which subsequently releases the phosphoramidate alkylator 20 to 500 μιη from the site of activation or reduction; or 20 to 100 μχη from the site of activation or reduction. Bystander effect of a phosphoramidate alkylator prodrug of ithe present invention can be measured using cellular spheroids and multilayer cellular assay (for example of such assays see Kyle et ah, Cancer Res. 2004, 64(17):6304-9 and West et al, Cancer Chemother. Pharmacol., 1987, 20(2): 109- 14); and as described in greater detail in Examples 35 and 37. Tumor cells can be grown in culture as multicellular spheroids to create an in vitro model of the tumor microenvironment in solid tumors containing'L hypoxic region and a quiescent cell population responding to the environmental stresses of,limited nutrients and increased waste production. These spheroids have the unique property of developing gradients of oxygen and nutrients as the aggregate of cells continue to divide and; grow outward. After the viable rim reaches approximately 150 μπι in size, a hypoxic region develops, that drives the cells in this region into a quiescent state and eventually to cell death. A necrotic core develops as a result of the dying cells. The spheroid can be divided into 4 distinct compartments for modeling the effectiveness of a hypoxic activated prodrug: 1) the outer aerobic and actively dividing region; 2) a region of intermediate hypoxia; 3) a region ofhypbxia where cells are not cycling; 4) and a necrotic core containing dead cells and cellular debris. The response of a drug will depend on a ■ ' " ' · ' ' ; 72 number of factors; the ability of compound to penetrate into the deepest regions of the spheroid. The activation of hypoxic activated prodrug (HAP) by nitroreductases; the reactivity of the activated drug i the cell in which it was activated; and the ability of the activated drug to leave the site from where it was activated and kill nearby cells (bystander ■ effect). The assessment of the effectiveness of a compound can therefore be evaluated on a number of different levels. The effect of the compound alone can be compared to cells in monolayer culture versus intact spheroids. The HAP can used as a monotherapy. The hypoxic fraction of the spheroid can be modulated by varying the concentration of <½ of the equilibrating gas and therefore change the ratio of the aerobic and hypoxic compartments. HA ' s can be combined with other chemotherapeutic agents that either target only the outer aerobic cells or are able to target the entire spheroid. The expected cell kill can be predicted by knowing the hypoxic fraction and the expected cell kill of .each of the monotherapies.

[0246] In one embodiment, the present invention provides a phosphorarnidate alkylator prodrug which upon activation such as bioreduction releases the phosphoramidate alkylator with a half life of less than 0.1 second; between 0.01 to 0.10 second, between 0.1 to 1.0 second, between 1.0 to 10.0 seconds, and between 10.0 to 100.0 seconds.

[0247] Anti cancers drags can bihd to tissue surrounding the vasculature and/or have high • molecular weights that impede diffusion and not reach in therapeutically effective concentrations hypoxic tumor zones that can be up to 150 - 200 μΜ away from the vasculature;.' In one embodiment, the present invention provides phosphoramidate alkylator prodrugs that can reach hypoxic cancer cells away from the vasculature. Some methods for determining the bystander effect are .described in greater detail in Examples 35 and 37. The phosphoramidate alkylator used in a hypoxia activated prodrug plays an important role to efficiently kill tumor i cells. For example,- for a hypoxia activated phosphoramidate alkylator prodrug, the cytoxicity of the phosphoramidate alkylator and its rate of cellular alkylation, and the cell membrane permeability of the prodrug and the phosphoramidate alkylator impact the hypoxic selectivity and hypoxic cytotoxicity of the phosphoramidate alkylator prodrug.

[0248] In one embodiment, the present invention provides phosphoramidate alkylator prodrugs that are safer than the corresponding phosphoramidate alkylators formed in vivo (at least ten and up to one million-fold safer.. In one embodiment, the increased safety results from a modification at the site of attachment of the Trigger T (activation of the phosphoramidate alkylator prodrug releases the alkylator/cytotoxic agent). In either event, the phosphoramidate alkylator prodrugs are converted into the corresponding alkylator in hypoxic tissues by virtue of the activation or reduction of the bioreductive group (Z3), resulting in its removal and the concomitant or subsequent release or generation of the- phbsphoramidate alkylator.

[0249] In one embodiment, the Trigger T is covalently bonded to the phosphoramidate alkylator, in a manner that masks or reduces the cytotoxic activity of the phosphoramidate alkylator. This masking effect can vary and can depend on the cytotoxic activity of (the phosphoramidate alkylator. Typically, the phosphoramidate alkylator prodrug will show at ' least about 10 fold less cytotoxic activity than the corresponding phosphoramidate alkylator, and can show up to about a million fold or less cytotoxic activity. In one version, the cytotoxic activity of the phosphoramidate alkylator prodrug is about 100 fold to about 10,000 fold less than the cytotoxic activity of the corresponding phosphoramidate alkylator. As one example, for a phosphoramidate alkylator with an IC50, IC90, or LC50 of 1 nM, the IC50, IC90, or LCso of the corresponding phosphoramidate alkylator prodrug can be 1 μΜ or greater.

[6250] In one version, compounds provided herein include as phosphoramidate alkylator prodrug, any phosphoramidate alkylator that can be linked to a Trigger T in a manner that yields a phosphoramidate alkylator prodrug that is at least about 10-fold to about 1 ,000,000- . fold, and topically about 100 to about 10,000-fold, less active as a cytotoxic agent than the corresponding phosphoramidate alkylator or modified phosphoramidate alkylator that is released from the compounds under hypoxic conditions.

[0251] To determine if a phosphoramidate alkylator prodrug is selectively active under anoxic or hypoxic conditions, cells are exposed to the drag either with air (normoxic) or ' without oxygen (anoxia) or with very little oxygen (hypoxia). One of skill in the art will recognize that cytotoxicity of a phosphoramidate alkylator prodrug as measured in an anti- - proliferation assay is expressed by the IC50; and,the cytotoxicity of a phosphoramidate alkylator prodrug as measured in a clonogenic survival experiment is expressed as IC]o or LCjo, IC90 or LC9o,,or IC99 or LC99. The^ratio of cytotoxicity, as measured for example by IC50, IC90, LC50, LC90, or LC99 determined in normoxia and hypoxia is called hypoxia cytotoxicity ratio (HCR) and can be a measure of the hypoxia selective cytotoxicity of the prodrugs of the present invention. The larger the HCR of the phosphoramidate alkylator prodrug the higher is its hypoxic cell selective toxicity and greater the hypoxic tumor killing ability of the prodrug relative to healthy nonnoxic cells. The HGR determined based on IC99 or LC99 is' larger than; that determined based on IC90 or LC90.

[0252] In a related embodiment, the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 50 μΜ and a HCR of 10 to 100,000! In a related embodiment, the phosphoramidate alkylator prodrug of the present invention has a . hypoxic cytotoxicity of 0.1 nM to 50 μΜ and a HCR of 25 to 100,000 (see EXAMPLE section). In another related embodiment, the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 5 μΜ and a HCR of 50 to 10,000 such as, for example, the compounds as described in Examples 29, 30 and 31. : [0253] In one embodiment, the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 5 to 1,000,000 folds more than the corresponding normoxic toxicity. In another embodiment, the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 10 to 10,000. folds more than the corresponding normoxic toxicity. In another embodiment, the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 25 to 5,000 folds more than the corresponding normoxic toxicity.

[0254] Tumors have a gradient of oxygen concentration that can vary from 10%, in tissues adjacent to ithe vasculature, to 0.5% in tissues about 150 μΜ away, and lower in tissues further away from the vasculature and near the necrotic core. In one embodiment, the present invention provides phosphoramidate alkylator prodrugs that can generate phosphoramidate alkylators, ,5-1 ,000,00; 10-10,00; and 25-5,000 folds more toxic than the corresponding prodrug, under a variety of oxygen concentrations. In one embodiment, the present invention provides phosphoramidate alkylator prodrugs generate phosphoramidate alkylators, 5- 1 ,000,00; 10-10,00; and 25-5,000 folds more toxic than the corresponding prodrug, under about 0.5-0.6% oxygen concentrations.

[0255] The logP of a phopsphramidate alkylator prodrug of the present invention can measure the lipophilicity or the hydrophilicity of the prodrug. In one embodiment, the present invention provides a phosphoramidate alkylator prodrug having a logP less, than 0. Such phosphoramidate alkylator prodrugs can be hydrophilic, such as a prodrug having formula XV wherein each Rn is H and can be easily formulated as an aqueous formulation for i.v. or i.p. injection. Another example of such prodrugs are compounds 24, 25 and 36.

[0256] In one embodiment, the present invention provides a phosphoramidate alkylator prodrug having a logP greater than 0. In one embodiment, the present invention provides a phosphoramidate alkylator prodrug having a logP between 0 and 4 such as those exemplified by formulas XIV; XX and XV wherein each Rn is methyl or, cyclopropyl, and administered in a patient can pass the cell membrane to penetrate inside cancer cells. Another example a prodrug having a logP between 0 and is 5, 6, 7, or 16. (for measured logP of phopsphramidate alkylator prodrugs of the present invention see EXAMPLES section). wherein ¾, R5j R7, and R8 are as defined as in formula (I). ~ (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci-C6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is→ or (iii) NR2R3 and NR4R5 both together are~"N ; (b) a Trigger-OH wherein Trigger is defined as in Formula (I), a trisubstituted phosphine, and (c) a dialkyl azodicarboxylate to yield the compound of formula: is selected from the group consisting of:

[0263] In; another embodiment, the reaction includes a solvent suc as ,THF, dioxane, a C C6 alkyl acetate, chloroform, dichloromethane, acetonitrile and the like. In another embodiment, each substituent in the trisubstituted phosphine is independently, selected from a Ci-C6 alkyl, CrC6 he'teroaIkyI, C3-C8 cycloalkyl, heterocyclyl, aryl, heteroaryl, and Ci-C6 alkoxy substituent., In another embodiment. Trigger T- is ' ■ wherein

[0264] In pother embodiment, the present invention provides a method to synthesize a ■ phosphoramidate alkylator prodrug comprising

[0265] (i) reacting in a solvent selected from THF, dioxane, dichloromethane, chloroform, ethyl acetate, propyl acetate, butyl acetate, or acetonitrile a compoumd of formula: wherein each ¾ i is independently hydrogen, cycloprpyl, methyl, ethyl, benzyl, or methoxy; each R9 is independently hydrogen, methyl, ethyl, propyl, or cyclopropyl; and ¾ is halo, methylsulforiyloxy, phenylsulfonyloxy, 4-methylphenylsulfonyloxy, and 4-halophenylsiilfonyloxy; (ii) a trisubstituted phosphine selected from hiphenylphosphine, tributylphospliine, tributylphosphite; and (iii) |di ethyl or diisopropyl azodicarboxylate; a method of synthesizing a WO 2007/002931 PCT/US200(')/025881 (i) reacting in an aprotic solvent, a Trigger-OH, wherein Trigger is defined as in Formula (I); a trisubstituted phosphine; and a dialkyl azodicarboxylate to yield an Intermediate (i); (ii) reacting the Intermediate (i) obtained from step (i) with a compound of formula a yield a dichlorophosphoramidate intermediate;

[0271] '(b) reacting the dichlorophosphoramidate intermediate in step (a) with a N-2- , ; haloethyl-N-(Ri3)ainmonium salt, wherein R!3 is hydrogen, Ci-Ce allcyl, C) -C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, heteroaryl, and a base in a solvent to yield a monochlorophosphoraniidate intermediate; and

[0272] (c) reacting the rrionochlorophosphoramidate intermediate obtained in step (b) with Trigger-OH and a base.in a solvent to yield the phosphoramidate alkylator prodrug.

[0273] In one embodiment, the dichlorophosphoramidate intermediate of step (a) is separated from the rest of the reaction mixture before subjecting it to the reaction in step (b). In another embodiment, the separation is performed by first removing excess POCI3 in vacuo and then distilling the dichlorophosphoramidate under reduced pressure. ;

[0274] In one embodiment, the phosphoramidate alkylator prodrug of step (c) is separated from the rest of the reaction mixture by flash column chromatography on silica gel. In one „ embodiment, the base employed in step (b) is a tertiary amine. Suitable tertiary amines employed in step (b) include trialkyl' amines, such as, triethyl amine or ' diisopropylethylamine. In one embodiment, the solvent employed in ste (b) is tetrahydrofuran (THF.) or dioxane.

[0275] In one embodiment, the monochlorophosphoramidate intermediate of step (b) is separated from the rest of the reaction mixture by flash column chromatography on silica gel before subjecting it to the reaction in step (c). In one embodiment, the base useful in step (c) is lithium, sodium, or potassiu n hexaalkyldisilazide; sodium or potassium hydride; . or lithium diisopropylamide. In one embodiment, the solvent employed in step (c) is dimethoxyethane, diglyme, diethylether or THF. —

[0276] In one embodiment, the present invention provides a method to synthesize a phosphoramidate alkylator prodiTig comprising the steps of:

[0277] (a) reacting in a solvent about 1 equivalent each of POCI3, a Trigger-OH^ and a base to yield a dichlorophosphate intermediate; and

[0278] (b) reacting the dichlorophosphate intermediate in step (a) with a N-2-haloethyl-N- (Ri3)amnionium salt, wherein R13 is hydrogen, C1-C6 alkyl, Ci-C6heteroalkyl, C3^Cg cycloalkyl, heterocyclyl, aryl, heteroaryl, and a base in a solvent to yield the phosphoramidate alkylator prodrug.

- ! .'' WO 2007/002931 PpT/US200f)/025881

[0279] In one embodiment, steps (a) and (b) are performed at temperatures below 0°C. In another embodiment, step (b) is performed at a temperature between 20-100 C higher than the temperature of step (a).

[0280] In another embodiment, the present invention provides a method, for synthesizing heterocyclic phopsphoramidate ankylator prodrugs of the present invention as shown below. wherein ¾ = Br or CI; e = 1 -3 ■ i . .

[0281] In one embodiment, the present invention provides a method to synthesize a phosphora hidate alkylator prodrug comprising the steps of:

[0282] (a) reacting PC13 with a N,N-di(2-hal0ethyl)ammonium salt and a base in a solvent to yield a monochlorophosphamide derivative;

[0283] (b) reacting the monochlorophosphamide derivative with Trigger-OH to yield an intennediate; and

[0284] (c) oxidizing the intennediate in step (b) to yield the phosphoramidate alkylator prodi-ug. I . (

[0285] .In one embodiment, the base used in step (b) is triethylarriine. In another embodiment, the solvent used in step (c) is dimethoxyethane, diglynie, or a C\-Ce alkyl acetate. In another embodiment, Trigger-OH is step (c) is :/ NO,

[0286] Various l-N-alkyl-2-aminoimidazole-5-carboxyIate can be synthesized as described schematically below: HCOOEt NHRCH2COQMe— -

[0287] The 1 -N-alkyl-2-aminoimidazole-5-carboxylates can be reduced to yield various 1 -N-alkyl-2-arnino-5-hydroxvinethylirnidazole derivatives employed in the present invention as bioreductiveigroup Z3.

[0288] The synthetic methods are provided in further detail in the EXAMPLES section below. : ■ ' i , . ,-■

[0289] Synthesis of bioreductive groups and phosphoramidate alkylator prodrugs, and methods of the present invention can be adapted from the references Matteucci et al., PCT Appl. Pub. No. WO 04/009667, and Hypoxia activated produgs US Pat. Appl. entitled.

"Hypoxia Activated anti-Cancer Agents"; deGroot et al, 2001 , Current Med. Chem. 8: 1093-1122; Denny et al, US Pat. Nos. 5,750,782; 5,780,585; 5,872,129; and 6,251,933; Davis et al, PCT Appl. Pub. Nos. WO 04/85421 and WO 04/85361 ; and Lin et al., US Pat. Appl. Pub. Nos. 2004/254103 and 2005/043244, and Borch et al., (supra).

[0290] Examples of methods to synthesize phosphoramidate alkylator prodrugs of the present invention are provided in further detail in the "EXAMPLES" section below.

Ilia. Metho'ds of Treatment

[0291] Iri one embodiment, the present invention provides a method of treating cancer in a patient in need of therapy thereof by administering to the patient a phosphoramidate alkylator prodrug of the present invention or one that is known. Known phosphoramidate alkylators are provided by the references Borch et al., supra. In one embodiment, the phosphoramidate alkylator prodrug employed in treating cancer according to the methods provided by the present invention has the formula selected from (I) - (XXVII). In one embodiment, the phosphoramidate- alkylator prodrug employed in treating cancer according to the methods provided by the present invention is selected from the compounds exemplified in the EXAMPLE section.

[0292] Cancer therapy with alkylating agents can lead to development of cancers that are resistant to these alkylating agents. Alkylating agents can l ill cancer cells in the more rapidly dividing or higher oxygen containing cancer region as compared to the cancer cells in the slower growing hypoxic cancer region. The latter cells survive the treatment by alkylators and can produce cells resistant to such alkylators. Increased activity of guanine-O6-Alkyltf ansferase, glutathione, glutathione transferases, the nucleotide excision repair pathway, and/or the mismatch repair proteins, and decreased permeation of actively transported drugs such as mechlorethamine and melphalan, are postulated to be responsible for cancer resistance to alkylators (for example, see, Hardman et ai, pages 1393 and 1433, supra). ■ ■

[0293] The prodrugs of the present invention are effective in treating cancers resistant to other therapies. Slowly dividing cancer cells in the hypoxic cancer zone act as a source of resistant cancer cells and strains and are killed by the prodrugs of the present invention; In one embodiment, the present invention provides a method of treating , a cancer resistant to treatment by one or more alkylators by adrninistering the compounds of the present invention alone or in combination with another anticancer agent. In one embodiment, a phosphoramidate alkylator prodrug of the invention is administered in combination with a drug having substantially no nephrotoxicity. In one embodiment the phosphoramidate alkylators prodrug is administered with carboplatin.

[0294] In jone embodiment, the present invehtibri provides phosphoramidate alkylators prodrugs which are not cross-resistant with known alkylators. In another' embodiment, present invention provides phosphoramidate alkylators prodrugs which are not cross-resistant ■with the alkylators cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustine, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and pxaliplatin. ■ ί

[0295] In. one embodiment, the present invention provides a method of treating cancer by administering as a first line therapy the compounds of the present invention alone or in combination with other anti-cancer agents. In another embodiment, the present invention provides a method of treating a metastatic cancer by administering as a first line therapy the compounds of the present invention alone or in combination with other anti-cancer agents. In one embodiment, the present invention provides a method of treating cancer by administering as a second line therapy the compounds of the present invention alone or in combination with other anti-cancer agents. In one embodiment, the present invention provides a method of treating cancer by administering as a third line therapy the compounds of the present invention alone or in combination with other anti-cancer agents, hi one embodiment, the present invention provides a method of treating cancer by administering after a prior treatment with surgery and/or radiation therapy the compounds of the present invention alone or in combination with other anti-cancer agents. In one embodiment, the present invention - 5 provides a method of treating cancer, the cancer having relapsed after prior chemotherapy, sugery, radiation or any combination of them, by administering the compounds of the present invention alone or in combination with other anti-cancer agents.

[0296] In methods for treating cancer provided by the present invention, an effective amount of phosphoramidate alkylator prodrugs is administered to the subject. Generally, the subject can be any -human or non-human mammal. The preferred subject is a human subject.

Other particular subjects include but are not limited to non-human primates, dogs, cats, farm animals and horses. In one version, the phosphoramidate alkylator prodrug is administered alone. In one version the phosphoramidate alkylator prodrug is administered in combination with one or more additional anti-cancer agents. In one version the phosphoramidate alkylator prodrug is administered in conjunction with a therapeutic cancer treatment, including but not limited to surgery and radiation. The phosphoramidate alkylator prodrug: will typically be administered in a pharmaceutical composition. Various pharmaceutical compositions that can be used {are described in the Formulations section infra.

[0297] The phosphoramidate alkylator prodrug and their'pharmaceutical compositions can 20 be used to treat any type of cancer in a subject, particularly in a human subject. Cancers that can be treated include but are not limited to leukemia, breast cancer, skin cancer, bone cancer, liver cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, ; : parathyroid^ thyroid, adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both' ulcerating and papillary type, metastatic 25 skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tunior, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, 30 . leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, and epidennoid carcinomas.

[0298]„ The phosphoramidate alkylator prodrug can particularly be used in the treatment of cancers containing significant areas of hypoxic tissue. Such cancers include but are not limited to lung cancer, especially non-small cell lung cancer, breast cancer, colon cancer, head and neck cancer, ovarian cancer, pancreatic cancer, and prostate cancer. Examples of types of cancers that can be treated with the phosphoramidate alkylator prodrugs of the invention are provided in the following references, each of which is incorporated in its entirety herin y reference Tidiriarsh et al., PCT Pat. Appl. No. PCT/US2005/047314 filed on 22 December 2005, and PCT Pat. Appl. entitled "Glufosfamide combination therapy", Attorney Docket No, 021305-005900PC; and US: Pat. App. No. 60/760,599 and 60/719,787 and PCT Pat. Pub. No. WO 2005/076888. Several of these cancers are discussed for illustrative purposes below. Those of skill in the art will appreciate that cancer chemotherapy often involves the simultaneous or successive administration of a variety of anti-cancer agents, and as discussed further below, a phosphoramidate alkylator prodrug can be used in combination therapies as provided by the methods described herein. Thus, in the description of illustrative cancers containing hypoxic regions amenable to treatment with a phosphoramidate alkylator prodrug, examples of combination therapies are also described.

[0299] Lung cancer affects more than 100,000 males and 50,000 females in the United States, most of who die within 1 year of diagnosis, making it the leading cause of cancer death. Current protocols for the treatment of lung cancer involve the integration of chemotherapy with or without radiotherapy or surgery. A phosphoramidate alkylator prodrug can be used as a single agent or in combination with existing combination therapies. A variety of combination chemotherapy regimens have been reported for small cell lung cancer, including the combinations consisting of cyclophosphamide, doxorubicin and vincristine (CAV); etoposide and cisplatin (VP-16); and cyclophosphamide, doxorubicin and VP-16 (CAVP- 16). Modest survival benefits from combination chemotherapy (etoposide plus cisplatin) treatment have been reported for non-small cell lung cancer.

[0300] Likewise, several different cytotoxic drugs have produced at least temporary regression; of ovarian cancer. The most active drugs in the treatment of ovarian cancer have been alkylating agents, including cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, cisplatin, and carboplatin. Current combination therapies for ovarian cancer include cisplatin or carboplatiri in combination with cyclophosphamide at 3- to'- 4-week intervals for six to eight cycles,. The compounds and methods described herein provide prodrug forms and methods for treating ovarian cancer in which a phosphoramidate alkylator prodrug as described herein is used as a single agent or in existing such combination therapy, either to replace an agent or in addition to the agent(s) currently used.

[0301] Cancer of the prostate is the most common malignancy in men in the United States and is the second most common cause of cancer death in men above age 55, and this cancer has been reported to consist primarily of hypoxic tissue. Several chemotherapy protocols have been reported for use in late stage disease following relapse after hormonal treatment. Agents for the treatment of prostate , cancer include the alkylators estramustine phosphate, prednimustine, and cisplatin. Combination chemotherapy is also used' to treat prostate cancer, including treatment with estramustine phosphate plus prednimustine and cisplatin, and 5-fluorouracil, melphalan, and hydroxyurea. The present invention provides methods for treating prostate cancer in which a phosphoramidate alkylator prodrug of the present invention is used in such combinations, either to replace an agent or in addition to the agent(s) currently used.

[0302] Cancer of the large bowel is the second most common cause of cancer death in the United States and'is likewise a cancer characterized by hypoxic regions. : While • chemotherapy in atients with advanced colorectal cancer has proven to ,be of only marginal benefit, 5-fluorouracil is the most effective treatment for this disease. 5-Fluorouracil is useful alone or in! combinatio with other drags; but is associated with only a 15 to 20 percent likelihood jof reducing measurable tumor masses by 50 percent or more. Using 5-FU in combination with the compounds and methods described herein, and the methods for treating colon cancer using a prodrug, can offer significant therapeutic benefit and potential for meeting the unmet need for better treatment methods for this disease.

[0303] In one version of the treatment methods, a phosphoramidate alkylator prodrug can . . j ■ :.■ - . ..■ · . '■:. ' '■' ' ''■ '■ '' " 1 - ' l - be used in various known approaches to cancer therapy including but not limited to "anti- } £ '■ - . ■ body-directed enzyme prodrug therapy" (ADEPT), "virus-directed enzyme prodrug therapy (YDEPT)i "gene-directed enzyme prodrug therapy" (GDEPT), and "bacteria-directed enzyme prodrug therapy" (BDEPT). The general uses of a phosphoramidate alkylator prodrug are not limited to' the foregoing treatment methods.

[0304]: n ahother aspect; the present invention provides a method of treatment of non-cancer hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation). In one embodiment, the hyperproliferative disease treated according to the present method is selected from the group consisting of allergic angiitis and granulomatosis (Churg-Strauss disease), asbestosis, asthma, atrophic gastritis, benign prostatic hyperplasia, bullous pemphigoid, coeliac disease, chronic bronchitis and chronic obstructive airway disease, chronic sinusitis, Crohn's disease, demyelinatihg neuropathies, dermatomyositis, eczema including atopic dermatitis, eustachean tube diseases, giant cell arteritis, graft rejection, hypersensitivity pneumonitis, hypersensitivity vasculitis (Henoch-Schonlein purpura), irritant dermatitis, inflammatory hemolytic anemia,, inflammatory neutropenia, inflammatory bowel disease, Kawasaki's disease, multiple sclerosis, myocarditis, myositis, nasal polyps, nasolacrimal duct diseases, neoplastic vasculitis, pancreatitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, periodoiital||disease, polycystic kidney disease, polyarteritis nodosa, polyangitis overlap syndrome, primary sclerosing cholangitis, rheumatoid arthritis, serum sickness, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, strictures of bilejducts, strictures (of duodenum, small bowel, and colon), silicosis and other forms of pneumoconiosis, type I diabetes, ulcerative colitis, ulcerative proctitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, vasculitis of the central nervous system, and Wegener's granulomatosis. t ■ . , ■ ■ · . , - [0305] In some embodiments of the invention, a compound of the present invention is administered to treat a hypei roliferative disease selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, restenosis, and benign prostatic hyperplasia.

In one embodiment, the hyperpriliferative disease treated is psoriasis, a disease characterized by the cellular hyperproliferation of keratinocytes. which builds up on the skin to form elevated, scaly lesions. In another embodiment, the hyperproliferative disease treated is multiple sclerosis, a disease.characterized by progressive demyelination in the brain. In another embodiment, the hyperproliferative diseases treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankylosis of joints affected. In another embodiment, the compounds of the present invention are administered to prevent a hyperproliferative disease resulting from cellular proliferation on a prosthesis implanted in a subject by coating the prosthesis with a composition containing a compound of the present invention. In another embodiment, the hyperproliferative disease • . .■ i. . : . ' . . '-92 ;■ ■ · .. . treated is benign prostatic hyperplasia, a disease in which prostate epithelial cells grow · abnormally and thereby block urine flow. '■li'Ib..Formulations, modes of administration, dosages . [0306] A phosphoramidate alkylator prodrug will typically be formulated as pharmaceutical formulations for administration to a subject. Described in this section are modes of administration, formulations, and dosages that can be used when treating cancers using a phosphoramidate alkylator prodrug described herein.

[0307] Administration of a phosphoramidate alkylator prodrug for the treatment of cancer can be effected by any method that enables delivery of the prodrugs to the site of action, the, hypoxic region of a tumor. Many cancer drugs are administered by intravenous injection, and a phosphoramidate alkylator prodrug can be formulated for such administration, including not only ready-for-injection formulations but also lyophilized or concentrated fonnulations that must be rehydrated of diluted, respectively, prior to injection. In addition to these formulations, a phosphoramidate alkylator prodrug can be formulated for administration by oral routes, ;intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal routes. Those of skill in the art will recognize that a phosphoramidate alkylator prodrug may be activated by bacteria in the gut. If such activation is not desired, then the practitioner can employ a route of administration or a formulation that results in absorption.of a phosphoramidate alkylator ί ■ . : prodrug prior to its entry into the large intestine or colon. The actual route of administration ' and corresponding formulation of the phosphoramidate alkylator prodrug will depend on the type of cancer being treated, the phosphoramidate alkylator prodrug selected for administration, the severity of the cancer, and the age, weight, and condition of the patient, . among other factors.

[0308] The amount of a phosphoramidate, alkylator prodrug administered, and thus the amount of 'the phosphoramidate alkylator prodrug contained in the dose administered and the product comprising that dose, will be dependent on the subject being treated, the severity of the cancer; localization of the cancer, the rate of administration, the disposition of the prodrug (e.g., molecular weight, solubility and hypoxic and normoxic cytotoxicity), the cytotoxic ί ' *·■"■' . ■ agent released by a phosphoramidate alkylator prodrug, and the discretion of the prescribing physician.; '.' ■ '■

[0309] In one embodiment, the present invention provides a method of cancer treatment in a patient wherein an effective dosage is typically in the range of about 0.001 to about 0.1 g per kg body weight, or about 0.1 to about 35 mg/lcg day in single or divided doses. For a 70 kg human, this would amount to about 0.05 to about 7 g/day, about 0.2 to about 2.5 g/day. In some instances, dosage evels below the lower limit of the aforesaid range can be more than adequate, while in other cases still larger doses can be employed without causing any harmful side effect; larger doses can also be divided into several small doses for administration throughout the day by infusion for an hour or continuously using a peripherally inserted central catheters (PICG line) and portable intravenous bag and pump. >

[0310] In one embodiment, the effective dose of a compound of the present invention for treatement of cancer, and other hyperproliferative diseases is in the range of about 0.1 to about 35 mg/kg/day; about 0.5 to about 20 mg/kg/day; about 0.5 to about 15 mg/kg/day; about 0.5 to about 10 mg/kg/day; about 0.5 to about 8 mg/kg/day; and about 1 to about 5 mg/kg/day in single or divided doses. In one embodiment, the effective dose of a compound of the present invention for treatement of cancer and other hyperproliferative diseases is in the range of about 2 to about 8 mg/kg day; about 2 to about 4 mg/kg/day; and about 2 mg/kg/day. in single or divided doses. In one embodiment, the effective dose of a compound of the present invention for treatement of cancer and other hyperproliferative diseases is in the range of about 0.25 to about 2 5 mg/kg/day; about 0.25 to about 1 mg/kg/day; and! about 0.25 to about 0.5 mg/kg/day in single or divided doses. In one embodiment, the dose is administered i.v. daily, either as a monotherapy (compound of the present invention alone) or in conjunction (combination) with standard of care therapies: In one embodiment, the effective dose for treatement of cancer and other hyperproliferative diseases is in the range, as described earlier administered once a week.

[0311] In one embodiment, a larger dose is administered intermittently (less frequently); a . dose in. the: range of about 3 to about 20 mg/kg; about 6 to about 10 mg/kg; or 8 mg kg is administered on once every three days for two weeks. In another embodiment, a dose in the range of about 5 to about 30 mg/kg; about 10 to about 15 mg/kg; or 12.5 mg/kg of the phosphoramidate alkylator prodrug is administered once a week for four weeks. In one embodiment, a dose'in the range of about 0.5 to about 8 mgkg/day is administered for 5 days over two yeekly cycles. , ,· ■. -.

[0312] In another ^embodiment, for treatment of human patients, the maximum daily dose of a. phosphoramidate alkylator prodrug is not greater than 500 mg/kg patient weight and, . ; Ί. : ' : . 94 " accordingly, a phosphoramidate' alkylator prodrug is administered in a daily dose in the range of about l mg of a phosphoramidate alkylator prodrug/kg of patient weight to about 500 mg of a phosphoramidate alkylator prodrug/kg of patient weight. In one embodiment, a phosphoramidate alkylator prodrug is administered in a daily dose in. the range of about 5 mg/kg to about 500 mg/kg"of the body weight of the patient to be treated. In another embodiment, the therapeutically effective dose is a daily dose of a phosphoramidate alkylator prodrug is about 10 mg/kg to about 250 mg kg of the body weight of the patient to be treated. I another embodiment, the therapeutically effective dose of a phosphoramidate alkylator prodrug is about 25 mg/kg to about 150 mg/kg of the body weight of the patient to be treated. In another embodiment, the therapeutically effective dose of a phosphoramidate alkylator prodrug is about 25 mg/kg to about 50 mg/kg of body weight of the patient to be treated. In another embodiment, the therapeutically effective dose of a phosphoramidate alkylator ' prodrug is about 1.25 mg/kg to about 12.5 mg/kg of body weight of the patient to be treated.

[0313] Guidance concerning administration can also be provided by and from studies in humans and other mammalian animals. A therapeutically effective dose determined for an animal can be converted to the corresponding human equivalent dose (HED) as described in the table below: aTb convert animal dose in mg kgto HED (assumes a 60 kg human) in mg/kg, divide animal dose by HED convertion factor. For species not listed or for weights outside the standard ranges, human equivalent dose (HED) can be calculated from the formula: HED = animal dose in mg/kg x (animal weight in kg/human weight in kg)0'33. b For example, cynomolgus,; rhesus, or stumptail.

[0314] To achieve therapeutic effectiveness, the therapeutically effective daily dose of a phosphoramidate alkylator prodrug is usually administered multiple times to the patient. In one embodiment, a phosphoramidate. alkylator prodrug is administered daily for a period of time. Typically, daily administration for at least 3 consecutive days will be employed. In related embodiments, administration is for at least 5 consecutive days, at least 7 consecutive ', days, or at least 10 consecutive days. Depending on the dose, formulation, and route of administration selected by the practitioner and the convenience of the patient, the entire daily dose can be. administered once daily, or the daily dose can be administered in multiple smaller doses through the course of a day (including by infusion with a pump or intravenous administration). For example, the dose can be divided into two smaller doses and administered twice daily, or divided into three smaller doses and admimstered thrice daily. It will be apparent to one of skill in the art of cancer treatment that, as used herein, "daily" administration is not limited to one administration per day but can include multiple administrations. ,

[0315] Administration schedules other than consecutive daily administration can also be used. Administration once every other day (qod) is particularly convenient, and administration once every third day, or once a week can be appropiate in some instances, but in any event, . a phosphoramidate alkylator prodrug is repeatedly administered over a period of time. For example, 'whether administration is daily (including, as noted, a divided daily dose), every other day, or less frequently, in one embodiment a phosphoramidate alkylator prodrug is administered at least 2 days per week for at least two, three, four, five or at least six consecutive weeks, or, alternatively, for at least two, three, four, five or at least six, weeks within a six-month period, or, alternatively, for at least two, three, four, five or at least six weeks witnin a twelve-month period. In-one embodiment; a phosphoramidate alkylator prodrug is?administered at least 3 days per week for at least two, three, four, five or at least six consecutive weeks, or, alternatively, for at least two, three, four, five or at least six weeks within a six-month period, or, alternatively, for at least two, three, four, ;five or at least six weeks within a twelve-month period. In one embodiment a phosphoramidate alkylator prodrug is administered at least 10 days per month, optionally at least 20 days per. month, for. at least one month or at least two, three, four,, five or at least six consecutive months, or, alternatively, at least one, two, three, four, five or at least six months in a 6-month period. [03.16] In one embodiment, the administration of the therapeutically effective dose is continued for multiple days, typically for at least three consecutive days, and often for at least ,-. · .■.■ ■ ' , . 96 five to ten consecutive days, or for a week, or for several weeks or more. Thus, a patient can be administered a phosphoramidate alkylator prodrug in accordance with the present methods for several days, a week, a month, two months, three months, six months, or a year or longer.

[0317]' Consistent with administration regimens of other anticancer agents, a phosphoramidate alkylator prodrug cantbe administered in multiple "rounds" of administration. For example, in some embodiments, a phosphoramidate alkylator prodrug can be administered once daily for at least three to ten, or at least five to ten consecutive days, and such three to ten or five to ten day treatments can be repeated once, twice, or three or more times, sometimes with a no-treatment (with a phosphoramidate alkylator prodrug) period ranging from one to several weeks between each multiple-day treatment. Similarly, in some embodiments, a phosphoramidate alkylator prodrug is administered every other day for two to ten administrations, more often three to ten administrations, or five to ten administrations, and such two, three or five to ten administrations qod can be repeated once, twice, or three or more times with a no-treatment (with a phosphoramidate alkylator prodrug) period ranging from one to several weeks between each multiple-day treatment. Other ■ multiple-round schedules for administration will be apparent to the skilled practitioner quided by this disclosure.

[0318] In one aspect, "administering a therapeutically effective dose or regimen of a ■ t ■ ■ ■ J phosphoramidate alkylator prodrug" refers to (i) administering a phosphoramidate alkylator prodrug in the ranges'stated (e.g., 1 mg to 1 g of a phosphoramidate alkylator prodrug per kg of patient wei ght, typically 25 to 150 mg of a phosphoramidate alkylator prodrug per kg of patient weight) for a specified minimum number of days within a specified time period, wherein the administration of a phosphoramidate alkylator prodrug has a therapeutic effect on the cancer in the patient. Illustrative therapeutically effective dose regimens for a phosphoramidate alkylator prodrug include those described herein, such ;as administration of a phosphoramidate alkylator prodrug for 3 consecutive days, 5 consecutive days, 7 consecutive days, 10 consecutive days, at least 3 days per week, at least 3 days per week for one month, at least 10 days per month, and at least 20 days per month.

[0319] i optimizing a phosphoramidate alkylator prodrug treatment regimen according to the present invention, the dose and frequency of a phosphoramidate alkylator prodrug administration can be selected to achieve a maximal sustained area under the plasma concentration curve (AUC) over the course of treatment. The theoretically optimal dosing * WO 2007/002931 PCT/US2006/025881 regimen will result in a maximal exposure of the tumor cells to a phosphoramidate alkylator prodrug, as measured by AUC, while minimizing the maximal plasma concentration (Cm.x) for any single administration. A higher Cmax will contribute to toxicity while the AUC will detennine efficacy. As is understood in the artJor other cancer therapeutic drugs, treatment with a phosphoramidate alkylator prodrug can be suspended temporarily if toxicity is observed, or for the convenience of the patient, without departing from the scope of the invention, and then resumed.

[0320] In one embodiment, the pharmacokinetics of the phosphoramidate alkylator prodrug of the. present invention employed for the treatment of cancer can determine the dose, the method of administration, and the kind of cancer that is treated with the phosphoramidate alkylator prodrug. In one embodiment, the phosphoramidate alkylatpr prodrug of the present mvention can have a in vivo half life of between 1 to 300 minutes.. In one. embodiment, the compounds 'of the present invention can have a in vivo half life of between 3 to 10 minutes. In one embodiment, the compounds of the present invention can have a in vivo half life of between, lOito 30 minutes. A short half life of the phosphoramidate alkylator prodrug can require an infusion time in treatment that is longer than that required for a phosphoramidate alkylator prodrug having a longer half life. A short half life of the phosphoramidate alkylator prodrug can increase the maximum tolerated dose (MTD) for that prodrug.

[0321] hi another embodiment, the present invention provides phosphoramidate alkylator ■ ' ■ I ■ - ; . .. · .. " · . ' prodrugs that remain up to 20% unchanged when incubated with mouse liver microsomal (update with human example and data if available) protein for 30 minutes. In another embodiment, the present invention provides phosphoramidate alkylator prodrugs that remain -80% unchanged when incubated with mouse liver microsomal protein for 30 minutes. In another embodiment, the present invention provides phosphoramidate alkylator prodrugs that remain greater than 80% unchanged iwhen incubated with mouse liver microsomal protein for minutes. In another embodiment, examples of phosphoramidate alkylator prodrugs of the present invention. which when incubated' with mouse liver microsomal protein for 30 minutes remain greater than 80% unchanged include 1 , 25, arid 36.· The higlier the MLM stability of a prodrug of the invention, its therapeutically effective dose and undesirable patient side effects will be lower. i1

[0322] In a related embodiment, the bioreductive group of the phosphoramidate alkylator prodrugs of the present invention upon reduction/activation in a hypoxic tumor zone form a WO 2007/002931 ' - ' - ' p'CT/US2006/025881 phosphoramidate alkylator- TM conjugate. The phosphoramidate alkylator ÷ TM conjugate can diffuse and reach other parts of the tumor or other tumors in the case of a metastatic disease. Various pharmacokinetic parameters such as volume of distribution under steady state (Vss), clearance (CL), area under curve (AUC), mouse liver microsomal stability; (MLM stability), plasma stability, and Cmax of phosphoramidate alkylator prodrugs of the present invention were measured and listed in the EXAMPLES section (see also Hardman et ah, supra).

[0323] In re-treatment regimens, the dose can be adjusted to reflect patient tolerance of the prior treatment. In any event, as toxicity is observed during repeat admimstration, dosing can be temporarily stopped as severe symptoms are observed. The period of temporary halting of administration (drug holiday) can be ended at the time when the first organ of toxicity no longer contains significant concentrations of a phosphoramidate alkylator prodrug or a phosphoramidate alkylator released therefrom (which can be measured or determined indirectly by cessation of symptoms). Therefore, an intermittent dosing period can be defined not only by specific days but individualized by drug holidays that are based on symptoms and normal organ clearance of a phosphoramidate alkylator prodrug or a phosphoramidate alkylators released therefrom.

[0324] A 'formulation of a phosphoramidate alkylator prodrug can, for example, be in a form suitable for oral administration as a tablet, capsule, pill powder, sustained release formulation,, solution, and suspension; for parenteral injection as a sterile solution, suspension or emulsion; for topical administration as ari ointment or cream; and for rectal administration as a suppository. A formulation of a phosphoramidate alkylator prodrug can be in unit dosage forms suitable for single administration of precise dosages and will typically include a conventional pharmaceutical carrier or excipient.

[0325] Suitable pharmaceutical carriers include inert, diluents or fillers, water and various organic solvents. The pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like. Thus for oral administration, tablets containing various excipients, such as citric acid can be employed together with various disintegrants, such as starch, alginic acid, and certain complex silicates, and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc can be used to prepare the tablet forms of formulations of a phosphoramidate alkylator prodrug described herein. Solid compositions of a similar type can be employed in soft and hard filled gelatin capsules. Preferred materials, therefore, include lactose or milk sugar 'and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the prodrug therein • can be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.

[0326] Exemplary parenteral administration forms include solutions or suspensions of a phosphorami'date alkylator prodrug in sterile aqueous solutions, for example, aqueous polyethylene; glycols, propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. -'[0327] Methods of preparing various pharmaceutical compositions with a specific amount . of active drug are known, or will be apparent, to those skilled in this art in view of this disclosure. For examples, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17ώ Edition (1984).

[0328] The methods of cancer treatment employing a phosphorainidate alkylator prodrug of the present invention are effective in killing the most difficult to kill cancer cells growing in the hypoxic; region of a tumor. Once released in the hypoxic region a phosphorainidate prodrug can diffuse from the hypoxic cells and kill the cancer cells in adjacent regions containing increasing populations of rapidly dividing cells. The hypoxic region acts as a drug-factory to produce within a tumor an alkylator for killing adjacent normoxic cancer cells leading to a higher concentration of the phosphorainidate alkylator within the tumor, relative . to normal tissues. The use of the prodrug, to generate the phosphorainidate alkylator within the tumor can reduce toxic side-effects arising due to normal cell toxicity. After cancer cell in the normoxic region of the tumor are destroyed, a hypoxic region can become normoxic and start to divide. At this point, such cells can be killed by the phosphorainidate alleviators generated from a phosphorainidate alkylator prodrug of this invention or those known, or by other anticancer agents or cytoxins administered in combination with the phosphorainidate alkylator prodrug, as described in the following section.

IIIc. Combination therapies

[0329] In accordance with the methods of the invention, a phosphorainidate alkylator prodrug can be co-adininistered in combination with other anti-cancer agents ("anticancer agent"). Without intending to be bound by any particular mechanism or effect, such co-administration can in some cases provide one or more of several advantages over known cancer therapies, such as, for example co-administration of a phosphoramidate alkylator prodrug and the anticancer agent has a synergistic effect on induction of cancer cell death. Co-administration provides a better therapeutic result than administration of the anticancer agent alone, e.g., greater alleviation or amelioration of one or more symptoms of the cancer, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, partial or- complete remission, prolonged survival or other beneficial therapeutic results.

[0330] The co-adnunistration of a phosphoramidate alkylator prodrug increases the sensitivit of cancer cells to the anticancer agent, allowing lower doses of the anticancer agent to be adminstered to the patient or allowing an anticancer agent to be used for treatment of cells otherwise resistent to the anticancer agent or otherwise refractory to treatment. While the known anti-cancer agents in general targets the rapidly dividing cells in the norrnoxic region,, the phosphoramidate alkylator prodrugs of the invention target the hypoxic cells in the regions of tumors that are not efficiently killed by the anticancer agent alone.

[0331] As used herein, a phosphoramidate alkylator prodrug is "co-administered" with another anticancer; agent (also referred to herein as, "Agent") when a phosphoramidate alkylator prodrug and Agent are administered as part of the same course of therapy. In one embodiment, a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, (i.e., the initiation of the other cancer therapy), and treatment with a phosphoramidate alkylator prodrug is continued throughout the course of administration of the Agent (i.e., the.course of the other therapy). In another embodiment, a phosphoramidate alkylator prodrug is administered after the initiation or completion of the other cancer therapy. In other embodiments, a phosphoramidate alkylator prodrug is first administered contemporaneously with the initiation of the other cancer therapy. See for example combination therapies as described in EXAMPLE section.

[0332] In one embodiment, a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, and treatment with a phosphoramidate alkylator prodrug is continued1 after the cessation of administration of the Agent. In one embodiment, a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, and treatment with' a . phosphoramidate alkylator prodrug is continued during part of the period of adrhinistration of the Agent. For certain drugs, such as certain topoisomerase inhibitors, a phosphoramidate alkylator prodrug administration can be initiated and completed prior to the administration of the second drug.

[0333] In the presence of oxygen, the radical anion formed upon the reduction of Z3 reacts with oxygenito yield superoxide and Z3. Superoxide is a cytotoxin and the production of superoxide in normoxic tissues car ead to unwanted side effects. In one embodiment, the present invention provides a phosphoramidate alkylator prodrug administered in combination with a chembprotective agent or a chemoprotectant. Chemoprotective agents protect healthy tissue from the toxic effects of anticancer drugs. In one embodiment, the chemoprotective agent is a thiol or a disulfide. In one embodiment, the chemoprotectant can reduce superoxide. In another embodiment, the the chemoprotectant can react with the "Michael-receptor" generated from a phosphoramidate alkylator prodrug and prevent "Michael-receptor" from reacting with proteins and nucleic acid (see below).

[0335] i one version of the method of treating cancer using the a phosphoramidate alkylator prodrug, a phosphoramidate alkylator prodrug is administered in combination with an effective amount of one or more chemotherapeutic agents, an effective amount of radiotherapy, an appropriate surgery procedure, or any combination of such additional I therapies. /

[0336] When a phosphoramidate alkylator prodrug is used in combination with one or more of the additional therapies, a phosphoramidate alkylator prodrug and additional therapy can be administered at the same time or can be administered separately. For example, if a phosphoramidate alkylator prodrug is administered with an additional chemotherapeutic agent, the two agents can be administered simultaneously or can be administered sequentially with some time between administrations. One of skill in the art will understand methods of ' administering the agents simultaneously and sequentially and possible time periods between administration. See for example combination therapies as described in the EXAMPLE section.

[0337] The Agents can be administered as the same or different formulations and can be administered via the same or different routes.

[0338] . Chemotherapeutic agents that can be used in combination with the a phosphoramidate alkylator prodrug of the invention include, but are not limited to, busulfan, impfosulfan, piposulfan, benzodepa, carboquone, 2-deoxy-D-glucose, lonidamine and analogs thereof (refrence apps), glufosfamide, gemcitibine, erlotiriib, meturedepa, uredepa, altretamine,;,imatinib, triemyte triethylenethiophosphoramide, trmemyloiomelamine, chlorambucil, chlornaphazine, estraniustine, ifosfamide, gefitinib, mechlorethainine, mechlorethamine oxide hydrochloride, melphalan,'novembichin, phenesterine, rednimustine, ti fosfamide, uracil mustard, carmustinej chlorozotocin, fotemustine, nimustine, ranirnustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(l), anthj-amycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycih, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-l-norleucine, mycophenolic acid, riogalamycin, olivomycin, peplomycin, plicamyciri, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin; ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptbpurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, caniiofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L- asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, defofamide, demecolcirie, diaziquone, elfornithine, elliptinium acetate, etoglucid, flutamide, gallium nitrate, hydroxyurea, interferori-alpha, interferon-beta, interferon-gainma, interleukin-2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2Tethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, P T/US2006/025881 erlotonib, tenyposide, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, urethan, vinblastine, cyclophosphamide, and. vincristine. Other chemotherapeutic agents that can be used include platinum, derivatives, including but not limited to cis platinum, carboplatin, and oxoplatin.

[0339] · In one version, a phosphoramidate alkylator prodrug described herein can be used in combination with an antiangeogenisis inhibitor including but not limited to Avastin and similar therapeutics. In one version of the combination treatment methods, a subject is treated with an antiangeogenisis inhibitor and subsequently treated with a phosphoramidate ' alkylator prodrug. In one version of the combination treatment methods, a subj ect is treated with an antiahgeogenisis inhibitor arid subsequeritly treated with a phosphoramidate alkylator prodrug with another chemotherapeutic agent, including but not limited to Cisplatin, and carboplatin. hi one version of these combination methods of treatment using an antiangeogenisis inhibitor, the method is used to treat breast cancer.

[0340] In another embodiment, a phosphoramidate alkylator prodrug is .administered with an anti-cance agent that acts, either directly or indirectly, to inhibit the epidermal growth factor or EGFR receptor. EGFR inhibitors suitable for coadministration with a phosphoramidate alkylator prodrug of the invention include gefitinib and erlotonib.

[0341] In another version, a phosphoramidate alkylator prodrug is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIF Ϊ a) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIF1 a levels. HIFl a inhibitors suitable for use in this version of the methods and compositions described herein include PI 3 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)-(lS,4S,10S,21R)-7-['(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8>7,6]-tricos-16-ene-3,6,9,19,22-pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333;' genistein; indanone; staurosporin; protein kinase- 1 (MEK-1) inhibitors such as PD98059 i(2'-amino-3,-methoxyflavone); PX-12 (1-methylpropyl 2-imidazolyl disulfide); pleurotin PX-478; quinoxaline 1 ,4-dioxides; sodium butyrate (NaB); sodium nitropurruside (SNP) and other NO donors; microtubule inhibitors such as novobiocin, panzem (2- . methoxyestradiol or 2-ME2), vincristines, taxanes, epotriilones, discodermolide, and pcT/iUsoe/esesjL derivatives of any of the foregoing; coumarins; barbituric and thiobarbituric acid analogs; camptothecins; and YC-1 , a compound described in Biochem. Pharmacol., 15 Apr 2001 , 67(8):947-954, incorporated herein by reference, and its derivatives.

[0342] In another version, a phosphoramidate alkylator prodrug is administered with an anti-angiogenic agent, including but not limited to anti-angiogenic agents selected from the group consisting of angiostatin, an agent that inhibits or otherwise antagonizes the action of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, endostatin, mterleukin, lavendustin A, medroxyprogesterone acetate, recombinant human platelet factor 4, Taxol, tecogalan, thalidomide, thrombospondin, TNP- 70, and Avastin. Other useful angiogenesis inhibitors for purposes of the combination therapies provided by the present methods and compositions described herein include Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen (Nalfon), indomethacin (Indocin), ketoprofe (Orudis, Oruvail), ketpralac (Toradol), oxaprozin (Daypro), nabumetone (Relafen), sulindac (Clinoril), tolmetin (Tplectin), rofecoxib (Vioxx), ibuprofen (Advil), naproxen (Aleve, Naprosyn), aspirin, and acetaminophen (Tylenol).

[0343] In addition, because pyruvic acid plays ah important role in angiogenesis, pyruvate mimics and glycolytic inhibitors like halopyruvates, including bromopynivate, can be used in combination with an anti-angiogenic compound and a phosphoramidate alkylator prodrug to treat cancer. In another version, a phosphoramidate alkylator prodrug is administered with an anti-angiogenic agent and another anti-cancer agent, including but not limited to a cytotoxic agent selected from the group consisting of alkylators, Cisplatin, Carboplatin, and inhibitors of microtubule assembly, to treat cancer.

[0344] In addition to the combination of a phosphoramidate alkylator prodrug with the Agents described above, the present methods and compositions described herein provides a variety of synergistic combinations of a phosphoramidate alkylator prodrug and other anticancer drugs. Those of skill in the art can readily determine the anti-cancer drugs that act "synergistically" with a phosphoramidate alkylator prodrug as described herein. For example, the reference Vendetti, "Relevance of Transplantable Animal-Tumor Systems to the Selection of New Agents for Clinical Trial," Pharmacological Basis of Cancer Chemotherapy, Williams and Wilkins, Baltimore, 1975, and Simpson Herren et al, 1985, "Evaluation of In Vivo Tumor Models for Predicting Clinical Activity for Anticancer Drugs," Proc. Am. Assoc. Cancer Res. 26: 330, each of which is incorporated herein by reference, describe methods to aid in the determination of whether two drugs act synergistically.

[0345] While synergy is not required for therapeutic benefit in accordance with the methods of described herein, in one embodiment, the present invention provides a method of cancer treatment, wherein there is synergy between a phosphoramidate alkylator prodrug and another anticancer agent. Two drugs can be said to possess therapeutic synergy if a combination dose regimen of the two drugs produces a significantly better tumor cell kill than the sum of the single Agents at optimal or maximum tolerated doses. The "degree of synergy" can be defined as net log of tumor cell kill by the optimum combination regimen minus net log of tumor cell kill by the optimal dose of the most active single Agent.

Differences in cell kill of greater than ten-fold (one log) are considered conclusively indicative of therapeutic synergy.

[0346] When a phosphoramidate alkylator prodrug is used with another; anti-cancer agent, a phosphoramidate alkylator prodrug will, at least in some embodiments, be administered prior to the initiation of therapy with the other drug or drugs and administration will typically be continued throughout the course of treatment with the other drug or drugs, h some embodiments, the drug co-administered with, a phosphoramidate alkylator prodrug will be delivered at! a lower dose, and optionally for longer periods, than would be the case in the absence of a phosphoramidate alkylator prodrug administration. Such "low dose" therapies can involve, for example, administering an anti-cancer drug, including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin, at a lower than approved dose and for a longer- period of time together with a phosphoramidate alkylator prodrug administered in accordance with the methods described herein.

[0347] These methods can be used to improve patient outcomes over currently practiced therapies by more effectively killing cancer cells or stopping growth of cancer cell as well as dmiinishirig unwanted side effects of the other therapy. When employed in combination with a phosphoramidate alkylator prodrug, the additional anti-cancer agent(s) is dosed using either the standard dosages employed for those Agents ( i.e., when used without a phosphoramidate alkylator prodrug) or are less than those standard dosages.

[0348] The administration of a phosphoramidate alkylator prodrug in; accordance with the ί ■ . · methods described herein can therefore allow the: hysician to treat cancer with existing (or later approved) drugs at lower doses (than currently used), thus ameliorating some or all of !" . ■ -. . 106 the toxic side1 effects of such drugs. The exact dosage for a given patient varies from patient to patient, depending on a number of factors including the drug combination employed, the particular disease being treated, and the condition and prior history of the patient, but can be determined using only the skill of the ordinarily skilled artisan in view of the teachings 5 herein. [0349) Specific dose regimens for known and approved chemotherapeutic agents or antineoplastic agents (i.e., the recommended effective dose) are known to physicians and are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, N.J 0 and/or are available from the Federal Drug Administration. Illustrative dosage regimens for ' ' certain anti-cancer drugs are also provided below.

[0350] Cancer drugs can be classified generally as alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo-oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, 5 nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors. In accordance with the methods described herein, a phosphoramidate alkylator prodrug can be co-administered with iany anti-cancer drug from any of these classes or can be administered prior, to or after treatment with any such drug or combination of such drugs. In 0 addition, a phosphoramidate alkylator prodrug can be administered in combination with a biologic therapy (e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies). Biologies used for treatment of cancer are known in the art and include, for example, trastuzumab (Herceptin), tositumomab and niI Tositumomab (Bexxar), rituximab (Rituxan).

[0351] Alkylators useful in the practice of the methods described herein include but are not limited to busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar); glufosfamide, melphalan, L-PAM (Alkeran) jdacarbazine (DTIC-Dome), and teinozolamide (Temodar). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an 0 alkylator to treat cancer. In one version, the cancer is chronic myelogenous leukemia, multiple myeloma, or anaplastic astrocytoma. · ■■ ■ ■ . !■ · . -: . . . 107 i . ■ ■

[0352] In one embodiment, the present invention provides a method of; treating cancer treatable by administering an alkylator by administering the phosphoramidate alkylator prodrugs of the present invention or those alone or in combination with at least another alkylator or a prodrug thereof. Alkylators, such as, for example, cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustin'e, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carbpplatin, and oxaliplatin, and types of cancers treated using any one of such alkylators alone or in combination with other anti cancer or chemoprotective agents are described for example in the reference Hardman et ah, (supra).

[0353] In one embodiment, the present invention provides a method of treating cancer by coadministering a phosphoramidate alkylator prodrug with at least the allcylator Cyclophosphamide, in the treatment of Stages III and IV malignant lymphomas, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian adenocarcinoma, ,. retinoblastoma, and carcinoma of the breast. Cyclophosphamide is administered for induction therapy in doses of 1500-1800 mg/m2 that are administered intravenously in divided doses over a period of three to five days; for maintenance therapy, 350-550 mg m are dministered every 7-10 days,- or 110-185 mg/m2 are administered intravenously twice weeldy. In 'accordance with the methods described herein, a phosphoramidate alkylator prodmg is co-administered with cyclosphosphamide administered at such doses or at lower doses and/or for a longer duration than normal for administration of Cyclosphosphamide alone. . , J , . ■ ' '

[0354] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention together with a cancer treatment ijegimen using at least the alkylator Mechlorethamine. For example, : Mechlorethamine is used in the combination chemotherapy regimen MOPP (mechlorethamine, Oncovin (vincristine), procarbazine, and prednisone) in patients with Hodgkin's disease and administered by intravenous bolus administration is doses 6mg/m on , days 1 and 8 of the 28 day cycles of each course of treatment.

[0355] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the allcylator Ifosfamide. Ifosfamide is used to treat pediatric and adult sarcomas, carcinomas of cervix and lung, and in combination with other drugs for germ cell ·. .- '■' ) ■ :.■ testicular cancer. Ifosfamide is used as part of the ICE (Ifosfamide, Carboplatin, and Etoposide) ans RICE (Rituxan and ICE) regimens for treating lymphomas (see Hardman et al., supra).■■<. .

[0356] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Glufosfamide. Glufosfamide is in the clinic for the treatment of.pancreatic cancer or Gemzar resistant pancreatic cancer. Glufosfamide can be used for treating breast cancer, Morbus Hodgkin, gastrointestinal tract cancer, or as part of the GCE (Glufosfamide, Carboplatin, and Etoposide) or RGCE (Rituxan arid GCE) regimen, for treating lymphomas. (Tidmarsh et al, PCT Pat. Appl. No. PCT/US2005/047314 filed on 22 December 2005, and PCT Pat. Appl, entitled "Glufosfamide combination therapy", Attorney Docket No. 021305-005900PC; and US Pat. App. No. 60/760,599 and 60/ 19,787 . and PCT Pat. Pub. No. WO 2005/076888, incorporated in their entirety herin by reference).

[0357] . In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment -■ regimen using at least an alkylator selected from the group consisting of ethylenimines and methylmelamines. In another embodiment, the ethylenimine is Triethylenemelamine or Thiotepa. ii ' ·

[0358] Thiotepa can be used to. treat adenocarcinomas of the breast, ovary, and bladder, ■ · · . · · ■ . malignant Jymphomas,;bronchiogenic carcinomas, and Wilms' rumor. Thiotepa was used at high doses jn combination chemotherapy with cyclophosphamide in patients with refractory malignancies treated with autologous bone transplantation and to treat a variety of cancers including bladder, ovarian, breast, lung, brain, and lymphomas (see, International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, 1975, 9 : 286, Lyon, France; International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, 1990, 50 : 415, Lyorf, France; and MEDLINEplus, 2003; Drug Infonnation: Thiotepa, National Library of Medicine). The methylmelamine Altretamine is used to treat advanced ovarian cancer after failure of first round therapies. ■

[0359] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the. invention with a cancer treatment regimen using at least the alkylator Melphalan, Chlorambucil, or Bendamustine. Melphalan • * · ■ · ' ' 109 . ■ ■· ■ : .■ ■ . : is used to treat multiple myolema and can be administered orally. Chlorambucil is used to " treat chronic lyphocytic leukemia and primary macroblobulinemia. Bendamustme, developed by Salmedix Inc. can be used to treat hematological malignancies, such as, for example, non-Hodgkin's lymphoma, chronis lymphocytic leukemia, and multiple myeloma.

[0360] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Busulfan. Busulfan is used to treat chronic granulocytic leukemia and chronic myelogenous leukemia. High doses of busulfan can be used i combination with Cyclophosphamide to treat patients with acute myelogenous leukemia before bone marrow transplantation.

[0361] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a nitrosourea alkylator. In another embodiment, the nitrosourea alkylator is Carmustine. Carmustine can be used to treat Hodgkin's disease, lymphomas, . myelomas, malignant astrocytomas, metastatic tumors of the brain, melanoma, and gastrointestinal tumors. In another embodiment, the nitrosourea is Streptozocin which is used to treat pancreatic islet cell carcinoma.

[0362] Inj.one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a triazene alkylator. In one embodiment, the triazene alkylator is Dacarbazine. Dacarbazine is used to treat malignant melanoma, Hodgkin's disease, and adult sarcoma.' In another embodiment, the triazene alkylator is Temozolomide. Temozolomide can be used to treat malignant gliomas.

[0363] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a platinum coordination complex alkylator. In one embodiment, the platinum coordination complex alkylator is Cisplatin. Cisplatin can be used to treat cancer of bladder, head and heck, endometrium, small cell carcinoma of the lung, and some neoplasms of childhood. Cisplatin alone or with cyclophosphamide is used to treat advanced ovarian . cancer. Combination chemotherapy of Cisplatin with Bleomycin, Etoposide,' and Vinblastine is used to jtreat advanced testicular cancer; and with one of Paclitaxel, Cyclophosphamide, or Doxorubicin to treat ovarian carcinoma. ,

[0364] . Anthracyclines useful in the practice of the methods described herein include but are not limited to, doxorubicin (Adriamycin, Doxil, Rubex), mitoxantrone ( ovantrone), idarubicin (Idamycin), valrubicin (Valstar), and epirubicin (EUence). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an anthracyciine to treat cancer. In one version, the cancer is acute nohlymptiocytic leukemia, Kaposi's sarcoma, prostate cancer, bladder.cancer, metastatic carcinoma of the ovary, and breast cancer. ' [0365] As one example the compound (8S, 10S)-10-[(3-Arnino-2,3,6-trideoxy-alpha.-L- lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9, 10-tetrahydro-6,8, 11 -trihydroxy-1 -methpxy-5,12- naphthacenedione, more commonly known as doxorubicin, is a cytotoxic anthracyciine antibiotic isolated from cultures of Streptomyces peucetius var. caesius. Doxorubicin has been used successfully to produce regression in disseminated neoplastic conditions such as acute lymphoblastic leukemia, acute myeloblastic leukemia, Wiim's tumor, neuroblastoma, soft tissue and bone sarcomas, breast carcinoma, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, lymphomas of both Hodgkin and non-Hodglcin types, bronchogenic carcinoma, and gastric carcinoma. Doxorubicin is typically administered in a dose in the'range of 30-75 mg/m2 as a single intravenous injection administered at 21-day intervals; weekly intravenous injection at doses of 20 mg m2; or 30 mg/m2 doses on each of, three successive days repeated every four Aveeks. In accordance with the-methods of the methods described herein, a phosphoramidate alkylator prodrug is co-administered starting prior to and continuing after the administration of doxorubicin at such doses (or at lower doses). Cyclic Anthracyciine cytotoxin prodrugs useful in the practice of the methods described herein are provided by the reference Matteuci et ah, PCT Patent Aplication No. US05/008161.

[0366] Antibiotics useful in the practice of the methods described herein include but are not limited to;dactinomycin, actinomycin D (Cosmegen), bleomycin (Blenoxane), dauiiorubicin, and daunomycin (Cerubidine, DanuoXome): In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an antibiotic to treat cancer. In one version, the cancer is a cancer selected from the group consisting of acute lymphocytic leukemia, other leukemias, and Kaposi's sarcoma. . .

[0367] Aromatase inhibitors useful in the practice of the methods described herein include but are not limited to anastrozole (Arimidex) and letroazole (Femara). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an aromatase inhibitor to treat cancer. In one version, the cancer is breast cancer.

[0368] Bisphosphonate inhibitors useful in the practice of the methods described herein include but are not limited to zoledrohate (Zometa). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a biphosphonate inhibitor to treat cancer. In one version, the cancer is a cancer selected from the group consisting of multiple myeloma, bone metastases from solid tumors, or prostate cancer.

[0369] Cyclo-oxygenase inhibitors useful in the practice of the methods described herein include but are not limited to celecoxib (Celebrex). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a cyclo-oxygenase inhibitor to treat cancer. In one version, the cancer is colon cancer or a pre-cancerous condition known as familial adenomatous polyposis.

[0370] Estrogen receptor modulators useful in the practice of the methods described herein include but are not limited to tamoxifen (Nolvadex) and fulvestrant (Faslodex). hi accordance with the methods described herein a phosphoramidate alkylator prodrug is coadministered with an estrogen receptor modulator to treat cancer. In one version, the cancer is breast cancer' or the treatment is administered to prevent the occurrence or reoccurrence of breast cancer.

[0371] Folate antagonists useful in the practice of the methods described herein include but are not limited to methotrexate and tremefrexate. In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a folate antagonist to treat cancer. In one version, the cancer is osteosarcoma.

[0372] As one example, the compound N-[4-[[(2,4-diamino-6-pteridinyl)methyl methylamino]benzoyl]-L-glutamic acid, commonly known as methotrexate, is an antifolate drug that has been used in the treatment of gestational choriocarcinoma and in the treatment of patients with chorioadenoma destruens and hydatiform mole. It is also useful in the treatment of advanced stages of malignant lymphoma and in the treatment of advanced cases of mycosis' fungoides. Methotrexate is administered as follows. Forichoriocarcinoma, intramuscular injections of doses of 15 to 30 mg are administered daily for a five-day course, such courses repeated as needed with rest period of one or more weeks interposed between courses of therapy. For leukemias, twice weekly intramuscular injections are administered in doses of 30 mg/m2. For mycosis fungoides, weekly intramuscular injections of doses of 50 g or, alternatively, of 25 mg are administered twice weekly. In accordance with the -.methods' described herein, a phosphoramidate alkylator prodrug is co-administered with methotrexate Administered at such doses (or at lower doses). 5-Methyl-6-[[(3j4,5-tj±methoxyphenyl)-armno]methyl]-2,4-qumazolmedian-ime (commonly known as trimetrexate) ;is another antifolate drug that can be co-administered with a phosphoramidate alkylator prodrug. ,

[0373] Inorganic arsenates useful in the practice of the methods described herein include but are not limited to arsenic trioxide (Trisenox). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an inorganic, arsenate to treat cancer. In one version, the cancer is refractory acute promyelocytic leukemia (APL).

[0374] Microtubule inhibitors (as used herein, a "microtubule inhibitor" is any agent that interferes with the assembly or disassembly of microtubules) useful in the/practice of the methods described herein include but are not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), docetaxel (Taxotere)', epothilone B or D or a derivative of either, and discodermolide or its derivatives. Tubulin binding anticancer drugs and prodrugs thereof which can be used in the practice of the methods of the present invention are provided in the reference Matteucci et al, PCT Patent Application No. PCT/US2005/042095; US Patent Applications entitled "Tubulin Binding Anti Cancer Agents and Prodrugs Thereof (Attorney Ref Nos. 021305-008500US, 021305- 008400US and 021305-004520US). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a microtubule inhibitor to treat cancer. In one version, the cancer is ovarian cancer, breast cancer, non-small cell lung cancer, Kaposi's sarcoma, and metastatic cancer of breast or ovary origin. As one example, the compound 22-oxp-vincaleukoblastine, also commonly known as vincristine, is an alkaloid obtained from the common periwinkle plant (Vinca rosea. Linn.) and is useful in the treatment of acute leukemia. It has also been shown to be useful in combination with other oncolytic agents in the treatment of Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma, rhabdomyosarcoma, neuroblastoma, and Wilm's tumor. Vincristine is administered in weekly intravenous doses of 2 mg/m2 for children and 1.4 mg/m2 for adults. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-administered with vincristine administered at such doses. In one version, a phosphoramidate alkylator prodrug is not administered prior to treatment with a microtubule inhibitor, such as a taxane, I WO 2007/0,02931 PCT/US2006/025881 but rather, adriiinistration of a phosphoramidate alkylator prodrug is administered simultaneously with or within a few days to a week after initiation of treatment with a microtubule inhibitor.

[0375] Modifiers useful in the practice of the methods described herein include but are not limited to Leucovorin (Wellcovorin), which is used with other drugs such as 5-fluorouracil to treat colorectal cancer. In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a modifier and another anti-cancer agent to treat cancer. In one version, the cancer is colon cancer. In one version, the modifier is a compound that increases the ability of a cell to take u glucose, including but not limited to. the compound N-hydroxyurea. N-hydroxyurea has been reported to enhance the ability of a cell to talce.up 2-deoxyglucose (see the reference Smith et al., 1999, Cancer Letters 141: 85, incorporated herein by reference), and administration of N-hydroxyurea at levels reported to increase 2-deoxyglucose uptake or to treat leukemia together with administration of 2-deoxyglucose and a phosphoramidate alkylator prodrug as described herein is one version of the therapeutic methods provided herein. In another such version, a phosphoramidate alkylator prodrug is co-administered with nitric oxide or a nitric oxide precursor, such as ah organic nitrite or a spennineNONOate, to treat cancer, as the latter compounds stimulate the uptake of glucose.

[0376] Nitrosoureas useful in the practice of the methods described herein include but are not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt). In accordance with the methods described lierein a phosphoramidate alkylator prodrug is co-administered with a nitrosourea to treat cancer, hi one version, the cancer is prostate cancer or glioblastoma, including recurrent glioblastoma multiforme.

[0377] Nucleoside analogs useful in the practice of the methods described herein include but are not limited to mercaptopurine, 6-MP (Purinethol); fluorouracil, 5-FU (Adrucil), thioguanirie, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Fludara), azacytidine (Vidaza), pentostatin (Nipent), cladribine!(Leustatin, 2-CdA),.gemcitabine (Gemzar), and capecitabinei(Xeloda). In accordance with the methods described herein a phosphoramidate alkylator prodrug is coadministered with a nucleoside analog to treat cancer. In one version, the cancer is B-cell lymphocytic leukemia (CLL), hairy cell leukemia, adenocarcinoma of the pancreas, ■ ' " t ■' r ' ; ! - . ; 114' - ·. , · metastatic breast cancer, non-small cell lung cancer, or metastatic colorectal carcinoma. As one example, the compound 5-fluoro-2,4(lH,3H)-pyrimidinedione, also commonly known as 5-fluorouracil, is an antimetabolite nucleoside analog effective in the palliative management of carcinoma!of the colon, rectum, breast, stomachy and pancreas in patient's who are considered incurable by surgical or other means. 5-Fluorouracil is administered in initial therapy in doses of 12 mg/m2 given intravenously once daily for 4 successive days with the daily dose not exceeding 800 mg. If no toxicity is observed at any time during the' course of theitherapy, 6 mg/lcg are given intravenously on the 6th, 8th, 10th, and 12th days. No therapy is given on the 5th, 7th, 9th, or 11th days. In poor risk patients or those who are not in an adequate nutritional state, a daily dose of 6 mg/kg is administered for three days, with the daily dose not exceeding 400 mg. If no toxicity is observed at any time during the treatment, 3•mg/kg.canjbe given on the 5th; 7th, and 9th days. No therapy is given on the 4th, 6th, or 8th days. A/sequence of injections on either schedule constitutes a course of therapy. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-administered with 5-FU administered at such doses or with the prodrug form Xeloda with correspondingly adjusted doses. As another example, the compound 2-amino-l, 7-dihydro-6H-purine-6-thione, also commonly known as 6-thioguanine, is a nucleoside analog effective in the therapy of acute non-pymphocytic leukemias. 6-Thioguanine is orally administered in doses of about 2 mg lcg of body weight per day. The total daily dose can :be given at one time. If after four weeks of dosage at this level there is no improvement, the dosage can be cautiously increased to 3 mg/kg/day. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-administered with 6-TG administered at such doses (or at lower doses); : ;

[0378] Osteoclast inhibitors useful in the practice of the methods described herein include but are not limited to.pamidronate (Aredia). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an osteoclast inhibitor to treat cancer. : In ' one version, the cancer is osteolytic bone metastases of breast cancer, and one or more additional anti-cancer agents are also co-administered with a phosphoramidate alkylator prodrug. . .. . .

[0350] filatinum compounds useful in the practice of the methods described herein include but are not limited to cisplatin (Platinol) and carboplatin (Paraplatin). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a platinum compound1 to treat cancer. In one version, the cancer is metastatic testicular cancer, metastatic ovarian cancer, ovarian carcinoma, and transitional cell bladder cancer. As one example, the compound cis-Diaminedichloroplatinum (II), commonly known as cisplatin, is .. useful in the palliative treatment of metastatic testicular and ovarian tumors, and for the . treatment of transitional cell bladder cancer which is not amenable to surgery or radiotherapy. Cisplatin, when used for advanced bladder cancer, is administered in intravenous injections of doses of 50-70 mg m2 once every three to four weeks. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-adrrunistered with cisplatin administered at these doses (or at lower doses). One or more additional anti-cancer agents can be co-administered with the platinum compound and a phosphoramidate alkylator prodrug. As one example, Platinol, Blenoxane, and Velbam can be co- administered with a phosphoramidate alkylator prodrug. As another example, Platinol and Adriamycin can be coadministered with a phosphoramidate alkylator prodrug.

[0351] Retinoids useful in the practice of the methods described herein include but are not • limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin), and bexarotene (Targretin). In accordance Jwith the methods described herein a phosphoramidate alkylator prodrug is coadministered with a retinoid to .treat cancer. In one version, the cancer is a cancer selected from the group consisting of APL, Kaposi's sarcoma, and T-cell lymphoma.

[0352] Topoisomerase 1 inhibitors useful in the practice of the methods described herein include but are not limited to topotecan (Hycamtin) and irihotecan (Camptostar). In ' accordance with the methods described herein a phosphoramidate. alkylator prodrug is coadministered with a topoisomerase 1 inhibitor to treat cancer. Topoisomerase inhibitors and prodrugs thereof useful in the practice of the methods of the present invention are provided in the reference Matteucci et al., PCT Patent Application No. PCT/US2005/041959. In one version, the cancer is metastatic carcinoma of the ovary, colon, or rectum, or small cell lung cancer. As noted above, however, in one version of the methods described herein, administration of a phosphoramidate alkylator prodrug either precedes or follows, or both, administration of a topoisomerase 1 inhibitor but is not administered concurrently therewith.

[0353] Topoisomerase 2 inhibitors useful in the practice of the methods described herein include but are not limited to etoposide, VP-16 (Vepesid), teniposide, VM-26 (Vurnon), and etoposideiphosphate (Etopophos). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a topoisomerase 2 inhibitor to treat cancer. In one version, the cancer is a cancer selected from the group consisting of refractory testicular rumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer. As noted above, however, in one version of the methods described herein, administration of a phosphoramidate alkylator prodrug either precedes or follows, or both, administration of a topoisomerase 2 inhibitor but is not administered concurrently therewith.

[0354] Tyrosine kinase inhibitors useful in the practice of the methods described herein include but aire not limited to imatinib (Gleevec). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a tyrosine kinase inhibitor to treat cancer. In one version, the cancer is CML or a metastatic or unresectable malignant gastrointestinal stromal tumor.

[0355] Lonidamine analogs useful in the practice of the present invention are provided in the Matteucci et' al. U.S. Pat. Appl. Nos. 11/346632; 60/764,427; 60/764,438; and applications entitled "Heterocyclic Lonidamine Analogs" (Attorney Docket No. 021305-007220US; 021305-007900US) and PCT Publication 'Nos. WO 2006/015191, WO 2006/015263 and WO 2006/01007IA2. . ' ' ' . ' [035.6] ..Tims, described herein are methods of treating cancer in which a phosphoramidate alkylator prodrug or a pharmaceutically acceptable. salt thereof and one or more additional anti-cancer agents are administered to a patient. Specific versions of such other anti-cancer agents include without limitation 5-methyl-6-[[(3,4,5-1rimemoxyphenyl)amino]-methyl]-2,4-quinazolinediamine or a pharmaceutically acceptable salt thereof, (8S,10S)-10-(3-amino-2,3,6-tridedxy-alpha-L-lyxo-hexopyi-anosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,l l-trihydroxy-l-methoxy-5,12-naphthacenedione or a pharmaceutically acceptable salt thereof; -.fiuoro-2,4(lH,3H)-pyrimidinedione or ^ pharmaceutically acceptable salt thereof; 2-amino- l,7-dihydro-6H-purine-6-thione or a pharmaceutically acceptable salt thereof; 22-oxo-vincaleukoblastine or a phaimaceutically acceptable salt thereof; 2-bis[(2-chloroethyl)amino]tetrahydro-2H-l,3,2-oxazaphosphorine, 2-oxide, or a phannaceutically acceptable;salt thereof; N-[4-[[(2,4-dianiino-6-pteridinyl)methyl]-methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof; or cisdiamminedichloro-platinum (II). . ■' 1 .

TV. EXAMPLES

[0357] In: the following examples, any reference to a compound designated by a letter is a reference to the structure shown next to or above that letter in the' corresponding reaction schemes..

Synthesis

[0358] : Methods to synthesize the phosphoramidate alkylator prodrugs of the present invention aire provided in section lib. Starring materials used in the synthesis of the; phosphoramidate alkylator prodrugs of the present invention were bought, when available, from commercial manufacturers, such as, for example, the Sigma-Aldrich Co. 1-N-Methyl-2-nitroimidazole-5-methanol was purchased from Syngene, India. Non-commercially available starting materials can be synthesized in via standard literature procedures. Such procedures can be identified via literature search tools such as SciFinder available from the American Chemical Society or Beilstein, available from MDL Software.

[0359] Reactions with moisture sensitive compounds, such as, for example, POCI3 and PCI3; and their mono and dichloro derivatives were performed employing anhydrous solvents and under nitrogen or argon. Separation of a product from the reaction mixture was performed employing a work-up where necessary, followed by vacuum distillation, crystallization, column chromatography, or preparative thick layer chromatography. A suitable eluent for the column chromatography of a compound can be determined by reading this disclosure and/or by determining the Rf of the compound by thin layer .chromatography and choosing a solvent which allows separation of the desired compound from undesired compounds. The choice of a particular eluent can depend, among other factors, on the polar nature of the compound, existence of other closely eluting compounds,: type of stationary phase such as silica gel or alumina used, and the amount of pressure used to elute the solvent through the stationary phase. In practice, different compositions of solvents can be .used to . separate the same compound.

[0360] Separated compounds were analyzed for their purity by standard analytical techniques, such as, TLC, NMR spectroscopy, and LC-MS, and stored in a freezer or a fridge, ayoiding moisture, light, or air. Stock solutions of phosphoramidate alkylator prodrug compounds were prepared in DMSO and stored in a freezer.

. . ' .■"■ Example 1 .

Synthesis of Compound 23 (TEA,.0,22 ml, 1.54 mmol). Temperature was increased to -30°C in one hour and then 2- chloroethylamine hydrochloride was added followed by TEA (1 ml, 7 mmol). After the temperature was raised to room temperature (rt), the( reaction was continued for one more hour, the reaction mixture was quenched with water and the organic layer was separated. The aqueous layer was extracted with DCM and the combined organic solution was dried and concentrated. Compound 23 was separated by flash column chromatography and analyzed by LC/MS and NMR spectroscopy to be pure.

,· Example 3 solution of sodium hydroxide (7g) in water (50 ml). The reaction mixture was stirred for 10 minutes, and extracted 4 times with ethyl acetate (75 ml). The combined organic layer was dried (MgS04) and evaporated to .give an orange oily residue. The residue was distilled in vacuo at 53-56°C (1 mm Hg) to yield an intermediate alcohol (5.94 g, 42% yield) as a clear, colorless liquid which was analyzed by LC/MS and 1 H NMR to be pure.

[0371] To a solution of the intermediate alcohol (3.7 g, 36.6 mmol) in dry THF (30 ml) a solution of HCl in dioxane (4.0M, 18:3 ml, 73.2 mmol) was added. The reaction mixture was cooled to 0°C and,SOCl2 (6.50g, 54.9 mmol) was added by syringe. The reaction mixture was refluxed (6 h), cooled, and concentrated to yield a residue. The residue was triturated with dry ether (100 ml), filtered, and residual volatiles removed in vacuo to yield 7i (5.42g, 95% yield) which was analyzed by *H NMR to be pure.

[0372] 7i (3.00g, 19.2 mmol) was added to POCl3 (15 ml) and refluxed under nitrogen for 7.5 hours. The reaction mixture was concentrated and the resulting oil distilled in vacuo through a short path distillation apparatus to yield 7ii as a clear, pale yellow oil (3.6g, 79% yield) which was analyzed by Ή NMR to be pure. [03,73] 7ii (0.50 g, 2.11 mmol) arid N-cyclopropyl-2-chloroethylamine hydrochloride (0.33 g, 2.11 mmol) were combined'in dry THF under argon. The reaction mixture was cooled to -78°C and DIEA (0.545 g, 4.22 mmol) added slowly by syringe, warmed to rt slowly, stirred for 1.5 hours and concentrated to give an orange oily residue. The residue was separated by flash chromatography over silica using 0 50% of hexane in ethyl acetate to give 315 mg (47% of theoretical) of pale yellow oil which was analyzed to be 7iii by MS.

[0374] N-methyl-2-nitroimidazole-5-methanol (76.8 mg, 0.489 mmol) was partially dissolved in dry THF (2 ml) under argon. The reaction mixture was cooled to -78°C and a solution of lithium bis(trimetliylsilyl)amide in THF (1.6M, 0.306 ml, 0.489 mmol) was added. After 15 minutes, a solution of.7iii (172 mg, 0.538 mmol) in 2 ml THF was added. After 15 minutes the reaction mixture was slowly warmed to rt, stirred for 2 hours, poured into 25 ml water and extracted 3 times with'ethyl. acetate (30 ml). The combined organic layers were dried over MgS04 and concentrated to give a yellow oily residue. The residue was separated by flash chromatography in 0-10% methanol in DC to yield compound_7 (110 mg, i51% yield) as a yellow oil which was analyzed by LC-MS and Ή NMR to be pure.

[0377] To a solution of N-methyl-2-nitroimidazole-5-methanol (180 mg, 1.14 mmol), triphenylphosphine.(300 mg, 1.14 mmol), and isophosphoramide mustard (lc, 127 mg, 0.57 mmol) in THF (10 ml) diisopropyl azodicarboxylate (DIAD, 0.22ml, 1.14 mmol) was added dropwise at rt. After two hours reaction mixture was concentrated and the residue separated by flash chromatography with 30-100% acetone in toluene yielding compound.36.

[0378] Compounds 23 and 26 were synthesized employing the procedure of Example 6.

[0379] N-methyl:2-nitroimidazole-5-methanol (50 mg, 0.318 mmol) was dissolved in dry THF (2 ml) under nitrogen. The solution was cooled to -78°C and a solution of lithium bis(trimethylsilyl)amide (1M in toluene, 0.35 ml, 0.35 mmol) was added by syringe. After 5 ' minutes a solution of bis(chloroethyl) phosphoramidic dichloride (91 mg, 0.35 mmol) in THF (2 ml) was added. After stirring at 78°C for 30 minutes, the temperature was reduced to - 20°C employing a,NaCl/ice bath and anhydrous ammonia was bubbled through the reaction mixture for? 5 minutes:. The reaction mixture was purged with nitrogen, warmed to rt, poured into 25 ml water and extracted with ethyl acetate (4 x 25 ml). The combined organic layers were dried (MgS04) and concentrated to give pale yellow oil which was separated by flash chromatography over silica gel using 0- 10% methanol in dichloromethane yielding compound! (32 mg,; 28 % yield) of an oil which soldified on standing and was analyzed by ' LC/MS and 1H NMR to be pure.

; Example 8 Synthesis of Compounds 25, 26

[0380] To a solution of 2-bronioethylammmonium bromide (1 .4 g) in DCM (90 mL) at - 10°C was added a solution of POCl3 (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14! mL) in DCM (25 mL) . The reaction mixture was filtered, the filtrate i concentrated to ca. 30% ofthe original volume and filtered. The residue was washed with .DCM (3x25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary ' evaporator, the resulting solution, chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

[0381] Isophosphoramide mustard can be synthesized employing the method provided in Example 8, 'substituting 2- bromo.ethylammmonium bromide with 2-chloroemylmiinimonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et ah, supra).

[0382] The phosphoramidate alkylator toxin: was transformed into compounds 2 Example 6 and the appropriate Trigger-OH.

! . Example 9 ' Synthesis of Compounds 37 - 105

[0383] The following compounds 37 - 105 were synthesized employing the Mitsunobu type coupling described for the synthesis of 25 or 36 above, and upon appropriate substitution of the Trigger-OH and the ifosfaniide mustard analog employed. For example, for the synthesis of compounds 40, 81, 83, 87, 89, 95, 96, 100, and 104, the ifosfamide mustard analog employed was H0P(=0)( HCH2CH2C1)2; in compounds 50, 53, 55, 56, 58 - 65, 6.8 - 71, 73 - 75, 77 -f 80, 82„ 84 - 86, 88, 90 - 92, 94, 97 - 99, 101 - 103, and 105; the ifosfamide mustard analog.employed is HOP(=0)(NHCH2CH2Br)2; in compounds 37, 39, 52, 54, and . 130 88 89 90 Examples 10 - 26 describes the synthesis of various Trigger-OH compounds employed in the synthesis of phosphoramidate alkylator prodrug's of the invention.

Example 10 Synthesis of Compound 52i , KOAc (190 mg, 1.92 mmol) in DMF (5 ml) was degassed thrice and PdCl2(dppf) (36 mg, 0.048 mmol) added to it at rt under an argon atmosphere. The reaction mixture was heated at 60°C for two hours,' diluted with ethyl acetate (EA) and washed with brine. The organic layer was dried, concentrated, and the residue separated by column chromatography on silica gel employing as eluent EA Hex (0 - 80%) to yield 52i. ed

[0388] To ajsolution of compound 68ii (100 mg, 0.31 mmol) and 3-amino-l-propanol , (0.047 ml, 0.62 mmol) in THF (2.5 ml), DIEA (0.162 ml, 0.93 mmol) was added at rt. The reaction mixture was stirred overnight and concentrated to yield a residue which was separated by column cliromatography on silica gel employing as eluent EA/Hex (0-80%) to yield compound 68i.

[0389] Compound 69i was made similarly as depicted in the scheme below. followed by Cul (8.5 rag, 0.043 mmol) and TEA (0.27 ml, 1.92 mmol), at rt, under argon atmosphere and the reaction mixture was heated at 60 °C for two hours. The reaction mixture was diluted with EA, washed with brine, the organic layer separated, dried, and concentrated to yield a residue which was'separated by column chromatography on silica gel employing as eluent EA\Hex (0-70%) to yield compound 58i. ; Example 17 64 64iii

[0398] 48% HBr (60 mL) was added dropwise to Example 18 Synthesis of Compound 2i

[0399] The vinyl derivative, 2iii, was synthesized according to the reference Cavalleri et al., J. Heti Chem., 1972, 9: 979, and oxjraercurated as follows. Hg(OAc)2 (208 mg, 0.653 mmol) was dissolved in water (0.7 mL) and THF (0.7 mL), followed by the addition of . compound 2iii (100 mg, 0.653 mmol). The reaction mixture was stirred at rt for 1.5 h, I. .

NaBKi (25 mg) added to it in portions, and after stining for 15 min the reaction poured into water, extracted with EA, the EA layer dried and concentrated to yield a residue which was separated by silica gel column chromatography employing as eluent EA/Hexane (0-100%) to yield com ound 2i (16 mg).

Example 19 Synthesis of Compound 94i

[0400] A solution of 94iii (7.1 g) in Ac20 (9.7 mL) was added dropwise into a solution of Fuming nitric acid (1.5 mL) was added into AcOH (12 mL) at 0 °C. The reaction mixture was warmed up to rt, stirred 1 r, fuming nitric acid (1 mL) was added dropwise into it and stirred for 1.5 h. The reaction mixture was poured into water, extracted with EA, the EA layer dried and concentrated to yield a residue which was separated by silica gel column chromato raphy employing as eluent EA/Hexane (0-100%) to yield compound 94ii.

Compound 94ii (600 nig, 1 .77 mmol) was suspended in methanol (10 mL) at 0°C followed by the addition of NaBH4 (141 mg) in portions into the reaction mixture over 5 min. NaB¾ (lOOmg) was added once every hour thrice, the reaction mixture was stirred for 3.5 h, poured into water, extracted with EA, the EA layer dried and concentrated to yield a residue which was separated by silica gel column chromatography employing as eluent EA/Hexane (0- 100%) to yield compound 94i (289 mg) as a yellow solid.

Example 20 Synthesis of Compound 96i

[0401] A mixture of A (1.4 g), CuCN (0.56g) and DMF (25 mL) was stirred at 140°C for 35 min and on 300 mL of crushed ice and stirred for 10 inin. The reaction mixture was then filtered and the residue was separated by column chromatography employing as eluent Hexane:EA ( 1 :0 to 2:3) to yield compound 96iii as yellow oil (617 mg). Compound 96iii was converted to alcohol 96i and separated by column chromatography, following a similar method as employed for compound 94iii in and using THF instead of MeOH as solvent in the reaction.' 01803014M7-01 13 188236/1 Example 21 Synthesis of Compound 99i 99ii 99iii 99i

[0402] A mixture of 99ii (500 mg), PdCl2(PPh3)2 (20S mg), and Cul (56.4 mg) was suspended in TEA (15 mL), the reaction mixture was degassed and flushed with Ar 6 times each. Propyne was bubbled through the reaction mixture for 15 nlin, and the reaction continued under a propyne atmosphere at 50°C bath for 2 h. The reaction mixture was poured into EA, filtered, the filtrate concentrated to yield a residue which was separated by 803014X17-01 139a silica gel column chromatography employing as eluent EA/Hexane (0-100%) to yield compound 99i (286 mg).

Example 22 Synthesis of 1 -N-methyl-2-amino imida2ole-5-carboxylic acid ethyl ester

[0403] Ethyl formate (500 mL) was added to sarcosin methyl ester hydrochloride (82 g, 585.7 mmol, grounded into powder prior to reaction) contained in a 1-L round-bottomed flask. The reaction mixture was cooled in an ice- ater bath,- stirred, a gas outlet connected with the flask, NaH (60%o oil suspension, 54 g, 1.35 mol) added slowly during a period of 2 h, and stirred at rt for about 14 h. Volatiles were removed using a rotary evaporator to yield a residue which was triturated twice with hexane (500 mL) to yield a sticky light brown paste which was dissolved in ethanol (400 mL) and cone HC1 (50 mL) and stirred at 110°C for 1.5 h. After the reaction mixture cooled down, the white precipitate was filtered off and the residue washed with 2 x 25 mL of ethanol. The filtrate was evaporated to yield a thick brown oil to which was added 10% aqueous HO Ac, H2NCN (45 g, 1.07 mol), and sodium acetate (88 g, 1.07 mol). The reaction mixture was stirred at 90-100°C for 1.5 h to yield a clear solution which was cooled, its pH adjusted to 1 using concentrated HC1 and the resulting solution concentrated to 1/5 its original volume using a rotary evaporator at a temperature not more than]l45°C. The concentrated reaction mixture was carefully neutralized by addition of 2C03 to & pH of 8-9 and extracted with EA (5 x 200 mL followed by 3 x 50 mL). The combined ethyl acetate layers were dried over MgS04,. filtered, and volatiles removed to yield 48 g:of 1 -N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester.

Example 23 (Synthesis of 1 -N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester i 140

[0404] Ethyl formate (850 mL) was added to sarcosine methyl ester HC1 salt (205 g, 1.46 ' mol, grounded into powder prior to use), potassium carbonate (205 g, 1.48 mol), and EtOH (800 mL,), stirred overnight at rt, and filtered. The filtrate was concentrated in a rotary evaporator during which the residue separated into two layers. The upper layer was separated and the lower layer was extracted with EA. Combined EA layers and the upper layer was dried over MgSC>4, filtered, and concentrated to yield 185 g (81%) of N-formyl sarcosine methyl ester which was used for the following reaction. NaH (60% oil suspension, 16.0 g, 0.4 mol) was carefully added in several portions in 1 h to a mixture of N-formyl sarcosine methyl ester (50 g, 0.34 mol) and ethyl formate (160 mL) cooled in an ice-water bath. The reaction mixture was stirred, the temperature raised to rt, and the stirring continued overnight. The reaction mixture was triturated twice with hexane (100 mL each time), the residue dissolved in EtOH (TOO mL) and concentrated HC1 (60 mL), and the reaction mixture stirred at 110°C. After 1 h, the reaction mixture was cooled down, filtered, the residue washed with EtOH and the filtrate( concentrated to yield a thick brown oil. The oil was added to 10% HOAc in water (200 mL)/NH2CN (35 g) and sodiuin acetate (90 g), stirred at 95°C. After lh the reaction mixture was concentrated to 1/3 its original volume in a rotary evaporator and its pH adjusted to about 9;by addition of sodium carbonate. The reaction mixture was then extracted with EA (8 x 100 mL), the combined EA layers dried, filtered, and concentrated to yield a residue which was purified by recrystallization to yield 1 -N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester ("amino ester").

Example 24 ; Synthesis of l-N-methyl-2-nitiOimidazole-5-carboxylis acid ethyl ester 141

[0405] A solution of the amino ester (36.94 g, 0.218 mol) in 200 ml of acetic acid was added drop wise to a solution of sodium nitrite (100 g, 1.449 mol) and water (300 ml) cooled in an ice-water bath, and stirred. The temperature of the reaction mixture, which was measured to be around -5 - 10°C was raised to rt and and the reaction mixture stirred overnight. The reaction mixture was extracted with DCM (3 x 150 mL). The combined DCM layers were dried and evaporated to yield a reddish residue which was separated by column chromatography on silica gel employing as eluent EA/hexahe (30%) to yield 1-N-methylr2-nitroimidazole-5-carboxylic acid ethyl ester ("nitro ester") as a light brown solid (27 g, yield 62%).

[0406] This method described in Example 24 and employing aqueous acetic acid is an improvement of the method using about 7% sulfuric acid (v/v) for the diazonium io formation from the amino ester. Using aqueous sulfuric acid, the reaction volume becomes large causing difficulty in stirring the reaction mixture effectively. For example, a reaction involving 150 g of the amino ester required a reaction mixture volume of about 12 L. The sticky nitro" ester formed as product in aqueous sulfuric acid and disrupted the stirring of the reaction mixture.

Example 25 Synthesis of l-N-methyl-2-nitt imidazole-5-carboxylis acid C02Et C02H

[0407] A suspension of the nitro ester (39.2 g, 196.9 mmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HG1 and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS04 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid ("nitro acid") as a light brown solid (32.2 g, 95%). 142 Example 26 Synthesis of 1 -N-methyl-2-nitroimidazole-5-carboxylis acid

[0408] A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 mL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetpnitrile bath (temperature < -20 °C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than 0°C. The reaction mixture was warmed up to 0°C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2-nitroimidazble-5 -methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

Example 27 ' Synthesis of Compound 119

[0409] To a suspension of l-N-methyl-2-nitroimidazole-5-methanol (50 nig, 0.32 mmol) in DME, LiN(TMS)2 was added at -78°C with vigorous stirring. After 10 min, compound 119i h, the y on silica Examples 28A-28V 143

[0410] Compounds 134 to 155 were synthesized by employing the corresponding substituted phosphormamidate and hydroxy substituted Trigger (Trigger-OH), according to the procedures described in Examples 1 -27 above.

Example 29A

[0411] The solubility of the following compounds is as listed below: Example 29B Antiproliferation Assay

[0412] To determine the effect of phosphoramidate alkylator prodrugs on cell proliferation, the antiproliferative activity of these compounds was tested in a multi-well Alamar Blue- based assay. Cell growth in the presence and absence of the test compound was compared, as measured by a fluorescence plate reader at excitation 550nm and emission 590nm (see Biosource International Inc., Tech Application Notes, Use of Ala ar Blue in the measurement of Cell Viability and Toxicity, Determining IC50). The following cell lines were tested with" 20,000 ο&\ΐ5/ν/ά]/500μ1 medium: NCI-H460 cells (ATCC HTB- 177, RPMI medium (Gibco Products, Invitrogen Corporation, Carlsbad, CA)), HT29 cells (ATCC HTB- 38, RPMI medium (Gibco)), MES-SA cells (ATCC CRL-1976, McCoy's 5a medium (ATCC)), MES-SA/Dx5 cells ((ATCC CRL-1977), McCoy's 5a medium (ATCC)), ACHN cells (ATCC CRL-1611, Minimum essential medium, Eagle (ATCC)), PC3 cells (ATCC CRL-1435, Ham's F12K medium (ATCC)). The cells were seeded in glass inserts placed in each well of a 24-well plate in the density and medium as specified above one day prior to compound testing. After 24 hours, these plates were divided into two groups - anoxia group and air group. A test compound was added to each well (200μί volume) in the treatment groups at concentrations varying from 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, to 0.01 μΜ. All test 144 compounds were serially diluted in complete medium with final DMSO concentrations less than or equal to 1% in each well. The cells in the anoxia treatment group were incubated for 2 hours in a Bactron II anaerobic chamber. The cells in the air treatment group were incubated for 2 hours in standard tissue-culture incubators. Following the 2 hour treatment with a test compound, the test compound was removed from each well, cells were washed with 500μ]1, medium, and incubated for 3 days in 500μΙ, fresh medium. After 3 days, cells were stained with 10% Alamar Blue for 2 hours after which the capacity of cells to proliferate was measured (as mentioned above), and the 50% growth inhibitory concentration (GI50 (also referred to IC50 herein)) of test compounds was calculated and tabulated in Table X below.

Table X: ICsn values (uM) 145 146 147 65 25/75 114 1 / 80 129 1/100 115 0.5/5 116 0.5/ 15 130 0.7720 66 0.3/100 67 48/>100 68 100/>100 69 71/>100 70 2/65 117 8/70 71 0.1/0.1 72 0.5/12 131 >100/ - >100 132 . 3/3 133 22/>100 104 0.4712 105 <0.1 / 1 Example 30 Antiproliferation Assay - Oxygen Dependence

[0413] Jo determine the oxygen dependence of phosphoramidate alkylator prodrugs, the antiproliferative activity of these compounds was tested in a multi-well Alamar Blue-based assay as previously described (see Example 29). NCI-H460 cells (ATCC HTB-177, RPMI medium (Gibco)) or HT29 (ATCC HTB-38, RPMI medium (Gibco)) were seeded at 20,000 οεΙΙβΛνεΠ/δΟΟμΙ, medium in glass inserts in 24-well plates one day prior to testing. The cells were incubated for 2 hours in a Bactron II anaerobic chamber flushed with gasses of the desired o ygen concentrations varying from anoxia, 0.1%, 0.3%, 0.6%; 1%, 10% oxygen, and air. The calculated IC5o values (μΜ) are tabulated in Table Yl (H460 cells) or Table Y2 (HT29 cells) below.

Table Yl : IC values (uM) in H460cells : 148 0.03 1 1 10 5 ' 40 1 30 60 60 >100 >100 0.3 10 10 100 >100 0.1 1 3 5 10 25 55 0.3 3 6 5 10 40 >100 >100 >100 >100 >100 0.007 0.85 >1 0.01 1 5 0.05 6 5 40 50 0 3 40 0.3 >100 >100 : 0.5 25 >100 0.03 0.5 0.2 0.5 . 0.4 45 >100 ; 0.1 0.7 4 1 >100 >100 : 0.3 10 15 0.3 3 25 ;i 45 >100 , 50 100 60 100 ' 0.006 0.01 0.02 ! 100 100 . , 3 3 0.5 2 1 7 7, : 10 20 10 t ; 0.5 1 ! 0.5 10 35 ! 1 30 . 60 149 55 5 8 12 56 0.5 8 10 123 ' >100 >100 >100 61 0.3 4 4 62 . 2 30 45 63 1 15 8 127 0.02 1 5 128 0.02 1 10 113 1 >100 >100 114 1 5 80 66 0.3 20 100 70 2 30 65 Table Υ2: IC50 values (μΜ) in ΗΤ29 cells Example 31 Clonogenic Assay - Oxygen Dependence

[0414] To determine the oxygen dependence of phosphoramidate alkylator prodrugs, a clonogenic survival assay was performed. Cells were plated in 60 mm glass dishes (5x105 cells per dish in 5mL of medium) 2 days prior to compound testing. The following cell lines were tested: NCI-H460 cells (ATCC HTB-177, RPMI medium (Gibco));iHT29 cells (ATCC HTB-38, RPMI medium (Gibco)), PC3 cells (ATCC CRL-1435, Ham's F12K medium (ATCC)). A solution of the test compound was made in complete medium immediately before the test and added directly to cells (2mL volume). Anoxia or hypoxia (less than 200ppm 02) was achieved by exposing the glass dishes in a Bactron II anaerobic chamber or in aluminum vessels (see Example 33) for 2 hours. For the anaerobic chamber, desired levels of oxygenation between 200ppm and air were achieved by flushing the anaerobic chamber with pre-calibrated gasses prior to experimentation. For the aluminum vessels, anoxia or 150 hypoxia wasiachieved by exposing the glass dishes in pre-warmed, air tight aluminum jigs to a series of five rapid evacuations and flushings with 95% nitrogen plus 5% carbon dioxide in a 37°C water bath on a shaking platform (controls are flushed as well). After the fifth evacuation and flushing, the platform (with water bath and jigs) was shaken for 5 minutes, after which one more evacuation and flushing were performed, and the jigs were transferred to a shaker in a 37 degree C incubator for the remainder of the 1 to 2 hour drug exposure. Levels of oxygenation between 200 ppm and air were achieved by varying the degree and number of evacuations. The oxygen concentrations in the medium and gas phases were checked using an oxygen electrode (Anima, Phoenixville, PA) in a specially modified aluminum jig that allowed for monitoring of both gas and liquid phases. Following the exposure to drug, the glass dishes were removed from the chamber or aluminum vessels and the drug was washed off the cells by rinsing with medium. The cells were then trypsinized and plated for clonogenic survival in plastic Petri dishes. Ten to 14 days later, the dishes were stained with crystal violet (0.25% in 95% ethanol), and colonies containing more than 50 cells were counted (see Example 33). The 90% growth inhibitory concentration (IC90, 90% killing, · 10% survival) of test compounds was calculated and tabulated in Table Y3 below.

Table Y3: IC90 values (μΜ) Example 32 151

[0415] To determine the electrochemical properties and reduction potentials of phosphoramidate alkylator prodrugs, cyclic voltammograms of these compounds were generated by Bio analytical Systems, Inc. All experiments were conducted with glassy carbon (3.0mm diameter) working electrodes, Ag/AgCl reference electrodes, and platinum wire auxiliary electrodes. Compounds were dissolved in lmL methanol to make final drug concentrations between 0.5 and 1.5mM after the addition of 9mL Phosphate Buffered Saline (PBS). The solution was added to an electrochemical cell vial and sparged with Argon for 5 minutes to remove most of the oxygen. Cyclic voltammetry was performed at 100 mV/sec and at 10,000 mV/sec scan rates at a glassy carbon working electrode. One test run was performed at a CGME mercury electrode (CGME in SMDE mode, 150μιη bore capillary, size 8 drop), but little difference was observed between mercury and glassy carbon voltammograms, so the mercury electrode was not used further. The single electron or multiple electron reduction potentials of compounds were generated at each scan rate and are tabulated in,the table below.

Table: Reduction Potentials (mV) Example 33 152 Clonogenic Survival Assay

[0416] The phosphoramidate alkylator prodrugs of the invention were tested in the assay as follows. Exponentially growing human H460 cells (obtained from the ATCC) were seeded . into 60mm notched glass plates at a density of between 2.5 and 5 xlO5 cells per plate and grown in RPMI medium supplemented with 10 % fetal bovine serum for 2 days prior to initiating drug treatment. On the day of the test, drug stocks of known concentrations were prepared in complete medium, and 2 ml of the desired stock added to each plate. To achieve complete equilibration between the surrounding gas phase and the liquid phase, the lid of the glass plate was removed and the plate shaken for 5 minutes on an orbital shaker. The plates were recovered and stored inside a glove-box. The glove-box was evacuated and gassed with either a certified anoxic gas mixture (95% nitrogen and 5% carbon dioxide) or with an aerobic (normoxic) gas mixture (95% air and 5% carbon dioxide). Cells were then incubated with the drug for 2 hours at 37°C.

[0417] At the end of prodrug treatment, plates were removed from each vessel, and the prodrug was promptly removed from the cells. Plates were washed with phosphate buffered saline and a solution of trypsin-EDTA and then trypsinized for 5 minutes at 37°C. Detached cells were neutralized: with medium plus serum and collected by centrifugation for 5 min at lOOxg. Cells were resuspended at approximately lxlO6 cells/ml and diluted 10 fold to yield stock concentrations for plating. The concentration of each stock was determined by counting with a Coulter Z2 particle counter. Known numbers of cells were plated, and the plates were placed in an incubator for between 7 and 10 days. Colonies were fixed and stained with a solution of 95% ethanol and 0.25% crystal violet. Colonies of greater, than 50 cells were counted, and the surviving fraction was determined.

[0418] HT 29 and cell based clonogenic assays were performed in the same way as described above and in Example 31.

[0419] Cytotoxicity of compounds (Tables 1A and IB) were determined in hypoxia and in normoxia by clonogenic assay employing H460. and HT29 cell lines as provided in Examples 31 and 33 and expressed as IC90 in 'μΜ, and by anti-proliferation assay performed by modifying a multi-well assay described by Hay et ah, J. Med. Chem., 2003, 46: 169-82 employing;H460, HT29, HCT116, and DX-5 cell lines and expressed as IC50 in μΜ (see Example 29). The ratio of IC50 or IC 0 determined in normoxia and hypoxia is called hypoxia 153 cytotoxicity ration (HCR) and can be a measure of the hypoxia selective cytotoxicity of the prodrugs of the present invention.

Table 1A 154 23 0.04 0.2 5 10 125 50 24 0.01 4 400 0.05 50 1000 26 0.1 35 350 27 2.5 100 40 31 83 >100 >1 32 50 100 2 34 <0.01 1.8 >180 0.075 50 625 36 -0.1 0.88 0.2 >100 >100 >110 >500 SI S2 Table IB P = Proliferation; C = Clonogenic; H = Hypoxia; N = Normoxic Example 34 Effect of Compound 25 on Cell Cycle Distribution

[0420] Cells (H60, PC3 and HT29) were seeded at a density of 1.0 x 106 cells/3ml medium per 60mm dish. After 24 h attachment, cells were exposed to Compound 25 at the indicated concentrations for 2 h under either normoxia (air) or anoxia (nitrogen). Cells were washed twice, and incubated for additional 22 h in fresh medium. Cells were trypsinized, centrifuged, and fixed in 75% ethanol at least for 24 h at -20°C. Cell cycle distribution was determined using Guava Cell Cycle reagent (Guava, Hayward, CA) by flow cytometry (Guava, Hayward, CA). The data demonstrate that Compound 25 induces cell cycle arrest in oxygen- and concentration-dependent manner in multiple human cancer cell lines.

H460 cells 156 WO 20()7/;ι002931 PCT/US2006/025881 Example 35 Spheroid Model

[0421] Two human cancer cell lines were used in these spheroid studies to determine the efficacy of the hypoxic activated phophoramidate alkylator prodrugs. HT29 colorectal adenocarcinoma (colon carcinoma) cells were seeded directly into a 125 ml spinner flask at 10,000 cells/ mL and grown in RPMI medium supplemented with 10% FBS and antibiotics. As these cells divided, they adhered to each other and formed spheroids. H460 lung carcinoma cells were seeded into a flask coated with a non-adherent surface to form small balls of cells that can be seeded into a spinner flask. To initiate H460 cell seeds, 150 cm2 tissue culture flasks were coated with 1% . agarose and then 10,000 cells per flask were added and allowed to grow in RPMI medium supplemented with 10% FBS and antibiotics for 3 to 5 days before seeding into spinner cultures. For both cell lines, growth medium was changed every day after the spheroids became visible to the eye.

[0422] Iii order to deteitnine the morphology and the location of hypoxic regions within an intact spheroid, whole spheroids were prepared for histology. For frozen sections, intact spheroids were washed in phosphate buffered saline (PBS) and embedded in OCT and rapidly frozen in a diy ice/2 -methylbutane solution before being stored at -80°C. For paraffin embedded sections, intact spheroids were fixed in a freshly prepared solution of 4% ί paraformaldehyde in PBS arid subsequently embedded and sectioned.

[0423] To assess the ability of a phosphoramidate alkylator prodrug to penetrate to the inner hypoxic cancer cells, become activated, release the phosphoramidate alkylator, and kill those inner cancer cells, the clonogenic. survival of spheroids exposed to drag for 2 h was measured. 157

[0424] Spheroids were placed in a new growth medium and incubated for at least 1 h before beginning experiments. Spheroids between 500 and 600 μιη were isolated by filtering the spheroid culture through a series of sterile mesh filters of defined size. Between 10 and 20 spheroids were placed on a. siliconized notched 60 mm Pyrex dish in 3 mL of medium with the desired concentration of the test compound. The dishes were placed in sealed aluminum vessels and exposed to a series of evacuations and gassings with certified gases containing 5% C02 and a defined amount of 02 (0% 02, 3% 02, 10% 02 or air). Spheroids were incubated in a shaking water bath to ensure both the equilibrium of the dissolved 02 in solution and the integrity of the spheroids in solution for 2 h. The test compound was removed and the spheroids were washed before being completely digested with trypsin. Since the necrotic core contains cellular debris a treatment with DNase I was required to yield a uniform single cell-suspension. Cells were resuspended at 106/mL and plated for clonogenic survival.

[0425] Initial dose response experiments were performed in monolayer cells under nitrogen, 0.6% 02, or. air to establish the appropriate dose range and the oxygen dependence of phosphoramidate alkylator release from a phosphoramidate all viator prodrug. Clonogenic survival was the end point and the data are summarized by the K¼o or C 0 values (the inhibitory concentration required to kill 90% of the cells and yield 10% survival).

Daunorubicin and cisplatin, each of which penetrates into speroids to a different extent, were employed to kill the outer aerobic cancer cells of the spheroid. Daunorubicin was used to penetrate the outer layers of a multicellular spheroid due to its high affinity toward cells and cisplatin was used at doses appropriate kill only the outer aerobic cancer cells. As a control for a bioreductive drag that killed cells under hypoxia in monolayer cultures, but not in multicellular cell culture due to its high reactivity and poor penetration, Tirapazamine was used both in monolayer based experments and in spheroids as tabulated below for H460 cells exposed for 2 h.

ICcio values for H460 cells exposed as monolayers or spheroids 158

[0426] A series of phosphoramidate alkylator prodrugs were tested in spheroids to determine their ability to penetrate into the inner lying hypoxic cancer cells, become activated, and kill the hypoxic cells. The results are tabulated below.

IC90 for H460 cells exposed as monolayers or spheroids to phosporamidate prodrugs for 2 h.

[0427] Similar results for the efficacy of Compound 25 were demonstrated in the HT29 spheroids as tabulated below:

[0428] The phosphoramidate alkylator prodrug was combined simultaneously with cisplatin or daunorubicin and the spreroids exposed for 2 li to the combination, followed by measurement of clonogenic survival. The results are tabulated below:

[0429] Phosphoramidate alkylator prodrugs demonstrate the ability to penetrate into the inner lying cells in the spheroid and kill hypoxic cancer cells alone and in combination with another agent that targets aerobic cancer cells.

I ■ . . · ' .

Example 36 V Antiproliferatioh Assay - DNA Mutant Repair Cells 159

[0430] Chinese hamster ovary cells mutant to specific DNA repair pathways were obtained from ATCC ' The following.cell lines were tested with 2,500 or 3,000 Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum and antibiotics: AA8 cells (ATCC CRL-1859), EM9 cells (ATCC CRL-1861), UV41 cells (ATCC CRL-1860), UV135 cells (ATCC CRL-1867), IRS1SF cells. All cell lines were initially screened with an anti-proliferation assay and those demonstrating sensitivity were retested with the clonogenic assay (as previously described) to confirm the proliferation results. Cells were exposed to selected doses of phosphoramidate alkylator prodrugs of the present invention for 2 h under hypoxic or aerobic conditions, the test compound was removed, and the cells assayed. The following table lists the cell lines, the pathway mutated, and the specific gene defect:

[0431] The following table lists the effect of exposure of various cell lines to Compounds 25 and 36 under anoxic or aerobic conditions and assayed by proliferation as measured by IC5o.

[0432] The following table lists the IC5o values for clonogenic survival for selected cells exposed to Comopund 25 under anoxic or aerobic conditions.

J

[0433] Only cell lines defective in homologous recombination were sensitive to Compound 25 under hypoxia. Since UV41 participates in both the nucleotide excision repair pathway as well as with the homologous recombination repair pathway, Compound>25 possibly also 160 produced a significant amount of monoadducts. However, UV135 which is also involved in nucleotide excision repair was not sensitive to Compound 25. The predominant lesions produced by ^Compound 25 were DNA interstrand crosslinks. These results were confirmed in UV41 and irsl SF cells with the clonogenic assay. The exposure under aerobic conditions produced the same spectrum of sensitivities as seen under hypoxia, indicating that the aerobic toxicity was also caused by DNA interstrand crosslink formation. Compound 36 exhibited a similar pattern of sensitivity in the mutant cell lines, indicating that Compound 36 also produced DNA interstrand crosslinks.

Example 37 Multilayered Cell Culture Assay

[0434] This example demonstrates the effect of Compound 25 on tissue penetration using multilayered cell culture (MCC) and to assess any bystander effect. MCCs were incubated with oxygenated media (20% 02 & 5% 02) or hypoxic media (approximately 0% 02) and the test compound was exposed from one side (exposed surface, normoxic side) while the other side was temporarily closed off (far side, hypoxic side). When MCC's are incubated in media at 20% 02 or 5% 02 a gradient in oxygen develops from the surface exposed to the media towards the far surface of the culture. The furthest 50 μη of tissue becomes depleted of oxygen. The extent of 02 depletion is greater with 5% than the 20% 02 gassed media; incubation with 5% 02 reflects the in vivo situation most closely. Incubating MCCs with media at 0% 02 models perfusion limited hypoxia,, where rumor blood vessels become completely depleted of oxygen and test compound must penetrate extensive distances to reach all cells. This situation therefore poses a greater barrier to ding penetration, if binding of activated drug acts to limit its penetration.

[0435] MCC based experiments were carried out with media gassed with 0, 5 or 20% 02 for 45 minutes prior to and during incubation with the test compound. HCT116 cells were grown to ajthickness. of 150μπι on a solid support and one side of the culture was clamped off to develop!diffusion limited hypoxia. Cultures were exposed to test compound for 1 In- under 0% 02, 5% 02 or 20% 02 and efficacy assessed by measuring the inhibition of BrdU incorporation. The cultures were incubated for a second hour in fresh media at 20% 02 and removed from the apparatus and returned to a normal growth chamber, where media flows over both sides of the MCC. Cultures were incubated for 24 hours prior to BrdUrd labeling and subsequent cryosectioning. BrdUrd labeling on the exposed and far sides of the MCC 161 :vere analyzed using immunohistochemical staining, microscope imaging and computer image analysis to assess the effect of Compound 25 on cell proliferation.

[0436] When cultures were exposed to graded doses of Compound 25 under 20% 02, 5 fold less compound was required on the far (hypoxic) side compared to the exposed (normoxic) side to produce comparable results, demonstrating penetration and hypoxic activation of Compound 25. When MCC's were exposed to test compound under a more physiologically relevant condition of 5% 02, Compound 25 was 10 fold more effective at inhibiting BrdU incorporation on the hypoxic side as compared to the normoxic side. Normoxic sides of cultures at 5% & 20% (¼ were equally affected by exposure to Compound 25.

[0437] Compound 25 is more effective on the hypoxic side of cultures under 5% 02 than with 0% 02. Comparison of normoxic versus hypoxic sides of cultures under 5% 02 demonstrated that Compound 25 penetrates effectively through relatively well oxygenated tissue. Compound 25 is capable of killing hypoxic cells located about 150μτη from functional blood vessels. Approximately 3 -fold reduction in exposure to Compound 25 to the hypoxic side was observed under 0% 02 relative to the exposure under 5% 02 conditions. Bystander effect was observed only at the highest concentration.

[0438] The following table lists the effect of e exposed to graded doses of Compound 25 as measured by IC50 (concentration to inhibit BrdU incorporation by 50%).

Example 38 ;. Metabolism of Compound 25 By Human and Mouse Microsomal Protein

[0439] n in vitro assessment of metabolic stability of a phosphoramidate alkylator prodrug (Compound 25) was performed using human (HLM), rat (RLM) and mouse (MLM) liver microsomal proteins containing cytochrome P450 enzymes. A solution of Compound 25 (500μL, 5μΜ) was prepared by diluting a DMSO stock solution 100 fold in a water.methanol bridge solution, adding microsomal protein (1 mg mL) in PBS/MgCl2, and enzymatic' reactions initiated by adding an NADPH solution. 50 μΐ of the reaction mixture was withdrawn at 0, 10, 20, and 30 minutes after addition of the NADPH solution, the 162 proteins were precipitated with acetonitrile and the clear supernatant was analyzed for the amount of Compound 25 by reversed phase LC-MS/MS. Nifedipine and testosterone were used as positive controls. The first study compared RLM to MLM (Table 1) and the second study compared HLM to RLM (Tables 2A and 2B) 163 min) 56 85 36 90 60 400 0.24 27.7 10.8 1.27 77.4 0.1S 44 26.1 Example 39 Iv Vivo Pharmacokinetics of Phosphoramidate Alkylator Prodrugs

[0440] Various plasma pharmacokinetic parameters of phosphoramidate alkylator prodrugs were determined in CD-I mice except where noted as listed below in Table 3.

Table 3 aBalb/c mice ! . Example 40 Iv Vivo Pharmacokinetics of Compound 25

[0441] Various plasma or tumor pharmacokinetic parameters of Compound 25 determined in CD-I mice except where noted as listed below in Table 4: Table 4 164 Saline 50 i.p. 30% PEG/70% 15.0 9.2 - - - Saline 50 i.v. 30% PEG/70% 2.0 27.5 177 10.1 - Saline 50- i.p. Saline 5.00 38.5 635 7.91 - 50 .o. Saline 15.0 0.93 40.4 25.7 13.6 i.p. 10% PEG 45.0 6.33 247 4.43 'Nude mice with H460 tumor "Tumor PK cBioavailabilitv Example 41 Cytochrome P450 Inhibition of the Metabolism of Compound 25

[0442] Eight reaction wells with 100 iL of a solution containing 50 mM potassium phosphate, pH 7.4, 2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 0.8 U/mL of glucose-6-phosphate dehydrogenase, andl:3 serial dilutions of the test compound (such as Compound 25) were prepared along with eight wells of 1 :3 serial dilutions of a suitable positive control inhibitor (such as furafylline for CYPl A2, sulfaphenazole for CYP2C9, N-benzylnirvanol for CYPC21 , quinidine for CYP2D6 and ketoconazole for CYP3A4). The concentrations of test compound ranges from 0.0229 μΜ to 200 μΜ. The reactions were initiated by adding 100 /zL of a pre- warmed enzyme/substrate solution. A zero time-point control reaction was prepared by adding 50 mL of 10% formic acid (400 mL of acetonitrile for 2C19) in water to 100 mL of cofactor solution to inactivate the enzymes, then adding 100 mL of enzyme/substrate solution. A control reaction with no inhibitor was also prepared. After a suitable incubation at 37 °C, the reactions were terminated by the addition of 50 mL of 10% formic acid in water (400 mL of acetonitrile for 2C19). The reactions were prepared and analyzed for the metabolite forms of the probe substrate (phenacetin for CYP 1 A2, diclofenac for CYP2C9, (S)-mephenytoin for CYPC219, dextromethorphan for CYP2D6 and midazolam^ testosterone and nifedipine for CYP3A4) using HPLC/MS/MS. Each assay was perfonned in duplicate. A summary of the IC50 values are listed below.

Table 5 165 2D6 0.21 >50 3A4 Midazolam 0.049 >50 3A4 Nifedipine 0.03 NI 3A4 Testosterone 0.10 >50 NI = No significant in libition detected Example 42 Determination of the Potential Metabolites of Compound 25 Formed in Mouse, Rat, Dog and Human Hepatocytes

[0443] Compound 25 is incubated with mouse, rat, dog, monkey and human cryopreserved hepatocytes at a concentration of 10 μΜ. The reactions are stopped at 0 (pre-incubation), 30, 60 and 120 minutes by quenching with acetonitrile prior to centrifugation and analysis by high-performance liquid chromatography (HPLC) in conjunction with tandem mass spectrometry (LC/MS/MS). Potential metabolites are identified by performing full scans from 100 to 520 amu. The product ion spectra of the potential metabolites are subsequently collected and compared to the product ion spectrum of the parent compound to determine whether each potential metabolite is related to Compound 25. The disappearance of the parent compound (Compound 25) and the appearance of potential metabolites over time are monitored by comparing the peak heights at each time point acquired.

Example 43 Determination of the In Vivo Pharmacokinetics of Compound 25 and its Metabolite (s) in Rat, Dog and Monkey

[0444] Phannacokinetic parameters of Compound 25 and its metabolite(s) in Sprague Dawley rats are determined following single intravenous administration of 5, 20, 50 and 100 mg/kg Compound 25. The pharmacokinetics of Compound 25 and its metabolite(s) will also be determined in beagle dogs and cynomologus monkeys following single intravenous administration of 20;mg/kg Compound 25. Concentrations of Compound 25 and its metabolite(s) in plasma are detennined by a LC/MS/MS method and mean phannacokinetic parameters are computed.

Example 44 Mass Balance Study in Rats 166

[0445] Nonnal and bile-cannulated Sprague-Dawley rats are administered 14C-Compound 25 as a single intravenous dose. Blood plasma, urine, feces and are collected at specified times and concentrations of total radioactivity are determined by liquid scintillation counting (LSC).

Example 45 Quantitative Whole Body Autoradiography

[0446] Sprague-Dawley rats are administered a single intravenous dose of 14C-Compound 25. At specified times, one rat per time point is euthanized. Blood is centrifuged to obtain plasma, and the blood and plasma are analyzed for concentration of radioactivity. Frozen rat carcasses are embedded in 2% CMC, frozen into a block and sectioned at 40 μτη in a Leica CM 3600 cryomicrotome. Collected sections are freeze-dried, mounted and exposed on phosphorimaging plates along with 14C autoradiographic standards for subsequent calibration of the image analysis software. Exposed screens are scanned using a Molecular Dynamics Storm 820 or 860. The concentration of radioactivity in select tissues including adipose (brown and white), adrenal gland, blood, brain (cerebrum, cerebellum, medulla) bone, bone marrow, cecum and contents, epididymis, esophagus, eyeball (Uveal tract, aqueous humor, lens), Harderian gland, heart, kidney (cortex, medulla, papilla and entire .section), large intestine and contents, liver, lung, lymph node submaxillary), pancreas, pituitary gland, prostate gland, salivary gland, seminal vesicles, skeletal muscle, skin, stomach (and contents), small intestine (and contents), spleen, spinal cord, trachea, thyroid and urinary bladder (and contents) are measured by image analysis. Autoradioluminographs and digital ΐ images are produced for each animal.

, Example 46 , Plasma Protein Binding of Compound 25

[0447] The protein binding in mouse, rat, dog, monkey and human plasma of Compound 25 is determined using ultrafiltration. Ultrafiltration is performed by aliquoting plasma spiked at three concentrations with Compound 25 into a Centrifiree® device in triplicate. All plasma samples are then equilibrated to 37°C. The Centrifree® apparatus is centrifuged at 37°C for 30 minutes at 2500 x g.. A 75 aliquot of the ultrafilitrate is spiked with the I.S. (deuterated Compound 25) and analysed using LC/MS/MS. The ultrafiltrates are analyzed and quantified using human ultrafiltrate standards for the calibration curve. » 167 Example 47

[0448] Example 47 demonstrates the usefulness of a compound of this invention in treating cancer employing a HT-29 human colon carcinoma xenograft mouse model.

[0449] Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line HT-29 was obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. Cells were maintained in a 37°C incubator with 5% C<¾. The HT-29 cells were harvested from culture and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of 10 mice was administered for three weeks, vehicle alone (saline and PEG; (lOmL/kg each), Group 1), Compound 36 alone (dissolved in 30% cyclodextrin in PBS) at a daily dose of 20, 60, or 200 mg/kg (Groups 2, 3 and 4, respectively), and .

Compound 36 at a daily dose of 20, 60, and 200 mg/kg given 2-3 hours after a dose of 10 mg/kg of 5FU (in saline) (Group 5, 6 and 7, respectively) and compared to a group receiving only 5FU at 10 mg/kg (Group 8) as tabulated below.

[0450] The body weight of each mouse was recorded twice per week. Growth of each xenograft was monitored by externally measuiing tumors in two dimensions using a digital caliper twice per week. Tumor volume (V) was determined by the following equation: V = (L x W2) 12, where L is the length and W is the width of a xenograft. Tumor volumes were measured twice weekly.

[0451] Administration of Compound 36 at 20, 60, and 200 mg/kg/day each reduced tumor growth compared to administration of vehicle alone. Administration of a combination of Compound 36 and 5FU resulted in greater and dose related inhibition of tumor growth comparedito vehicle, ha addition combinations of 60 and 200 mg/kg of Compound 36 reduced tumor growth to a greater degree than 5FU alone. 168 7 . . 200 + 5FU 58.2 23 8 5FU 38.7 -

[0452] Associated with these anti-tumor effects, there was some degree of weight loss and occasional mortality, particularly in the group treated with the high dose of Compound 36 but in other groups as well. Overall, Compound 36 showed varying rates of tumor growth inhibition.

Example 48

[0453] Example 48 demonstrates the usefulness of a compound of this invention in treating cancer employing a NCI H460, human colon carcinoma xenograft mouse model.

[0454] Female CB 17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, arid handled under pathogen-free conditions. Human colon carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested according to the procedure described in Example 47 and inoculated at 1 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of mice was administered for three weeks, as tabulated in the table below: Compound 25 (2.5 mg/ml in 10% PEG; administration route - i.p.) and Taxol (1 mg/ml in 5%ΈίΟΗ, ;5% Cremophor and 90% saline; administration - i.v. 2 h after administration of Compound 25). The body weight and tumor volumes were measured as described in Example 47 above.

Treatment protocol 169 Taxol 10 (q2d x 3) /week x 2 weeks Groups la and lb, n = 5; qld/qd = every day; q2d = every second day; q7d = every seventh day.

[0455] Results are presented in Table X2 based on tumor volume measurement on day 29 when vehicle treated mice had reached a volume of 946 mm3. Groups of 5 mice receiving saline or no treatment were added in order to indicate any vehicle effects but are not used for comparisons1 in this analysis.

[0456] The results demonstrate that all three regimens for dosing Compound 25 provided similar degrees of tumor growth inhibition and that combination therapy, particularly with every day dosing provided additional benefit. Each combination therapy was associated with some degree of weight loss but not large enough to cause any mortality. Overall the results indicate that Compound 25 is efficacious in this model of lung cancer arid provides additional benefit to that provided by the standard chemotherapeutic agent, taxol.

[0457] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective dose of about 2 to about 8 mg kg/day, for the treatment of cancer, particularly lung cancer, alone or in combination with Taxol™, wherein the daily dose can be administered with a decreasing frequency of dosing for higher doses compared to lower doses. , , Example 49

[0458] Example 49 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a H460, non-small lung carcinoma xenograft mouse model. Female CB17/SCED mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under 170 pathogen-free conditions. Human non-small lung carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 above, and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day S), each group of mice (ten per group) was administered for three weeks, as tabulated in the table below: Compound 25 (2.5mg/ml in 10% PEG; administration route - i.p.); Compound 24 (0.3, O. lmg/ml in 10%PEG, adininistration route - i.p.) and Taxol (1 mg/ml in 5% EtOH, 5% Cremophor and 90% saline; administration - z'.v. 2 h after administration of the test compound).

Treatment protocol * - 50% PEG [04S9] The body weight and tumor volume was determined as described in Example 47 above. Results for tumor growth inhibition measured on day 27 are as tabulated below. Comparisons were made on day 27 because that was the last day of measurements for the vehicle group and those animals were sacrificed.

[0460] These results demonstrate that daily doses of 3 mg/kg of Compound 24 and 25 mg/kg of Compound 25 inhibited tumor growth and that Compound 25 had a slightly greater benefit both as monotherapy and in combination with taxol. These effects were accompanied by mild weight reductions, particularly in the Compound 25 + taxol group. 171

[0461] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective dose of 2 mg/kg/day, for the treatment of cancer, particularly lung cancer, alone or in combination with Taxol™, and Compound 24 can be administered at a therapeutically effective dose of 0.25 mg/kg/day, for the treatment of cancer, particularly lung cancer, alone or in combination with Taxol™.

Example 50

[0462] Example 50 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a HT-29, human colon carcinoma xenograft mouse model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line HT29 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 above, and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of mice (ten per group) was administered for three weeks, as tabulated in the table below: Compound 24 (in %PEG), [administration route - i.p., administrated 2 h before 5-FU or cisplatin (CDDP; in saline) on the days the combination therapy was scheduled; 5FU alone (in saline), or CDDP alone.

Treatment protocol *- tumor location flank; ** - tumor location peritoneum; Q3d = every third day 172

[0463] In control groups, tumors were implanted in two locations as part of separate study of effect of location on control group tumor growth. These results had no impact on the interpretation of the study and all treatments were compared to the vehicle group with tumors on the same are of the body. The body weight and tumor volume were measured as described in Example 47. Tumor growth inhibition measured on day 25 when vehicle tumors had reached the maximal size and animals in that group were sacrificed is tabulated below

[0464] The results demonstrate that Compound 24 as monotherapy resulted in tumor growth inhibition of slightly more than 40% whereas combining Compound 24 administered in combination with CDDP or 5FU provided about 50-70% growth inhibition. According to this Example, the most therapeutically effective combination was that of Compound 24 and 5FU. The effects on tumor growth were associated with minor decreases in weights of the mice during treatment; however the mice recovered the lost weight after the end of treatment.

[0465] Using the mouse to HED conversion, Compound 24 can be administered at a therapeutically effective dose of about 0.25 to about 0.50 mg/kg/day, for the treatment of cancer, particularly colon cancer, alone or in combination with 5FU or CDDP.

Example 51

[0466] Example 51 describes the usefulness of a compound of this invention in treating i cancer as demonstrated employing a H460, non-small lung carcinoma xenograft mouse model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human non-small cell lung carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 above, and inoculated at 3 x 106 cells/ animal in the • 173 peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3, treatment was initiated in which groups of 10 mice received vehicle (Group 1), CDDP at 3 or 6 mg kg (Groups 2 and 3, respectively, IV one time), Compound 25 at 50'mg/kg in saline 5 times per week for two weeks (Group 4), Compound 25 at 100 mg/kg every three days for 5 times (Group 5) or the combination of each dose of Compound 25 with either 3 or 6 mg kg of CDDP (Groups 6 and 7, respectively). Results for groups receiving 50 mg/kg of Compound 25 are illustrated in the Figure 1. Figure 2 shows similar results for 100 mg kg of Compound 25.

[0467] These results performed with a saline formulated version of Compound 25 , demonstrate significant dose related decrease in tumor volume and increase in tumor growth delay with a daily dose of 50 mg/kg, and 100 mg/kg with less frequent dosing compared to that employed for the 50 mg kg daily dose. These data also demonstrate that both dosing regimens add to the effects of CDDP in this model.

[0468] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective doses of about 4 to about 8 mg/kg/day, for the treatment of cancer, particularly lung cancer, alone or in combination with 5FU or CDDP, wherein the daily dose can be administered with a decreasing frequency of dosing for higher doses compared to lower doses.

. Example 52

[0469] Example 52 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a HT-29, human colon carcinoma xenograft mouse model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line HT29 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm (day 8), each group of mice (ten per group) was administered for three weeks, as tabulated in the table below: Compound 25 in saline, administration route - i.p., administrated 2 h before CDDP on the days the combination therapy is scheduled and CDDP (in saline, IV).

Treatment protocol 174

[0470] The body weight and tumor volume was determined as described in Example 47. Data are based on tumor volumes at day 25 when tumors in the vehicle group had reached sufficient size to require that the mice be sacrificed. The results of inhibition of tumor growth are tabulated below.

[0471] The results demonstrate that monotherapy administering Compound 25, fonnulated in saline, at 50mg/kg day and 100 mg/kg/day with a variety of dose regimens results in inhibition of tumor growth in this model of colon cancer, and that treatment combination of Compound 25 and CDDP enlianced the effectiveness of Compound 25 for treatment of colon cancer in this model. These effects were accompanied by modest body weight loss, more so •in the combination groups; the mice recovered the lost body weights after the treatment ended. 175

[0472] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective doses of about 4 to about 8 mg/kg/day, for the treatment of cancer, particularly nonrsmall cell lung cancer, alone or in combination with CDDP, wherein the daily dose can be administered with a decreasing frequency of dosing for higher doses compared to lower doses.

Example 53

[0473] Example 53 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a H460, non-small lung carcinoma xenograft mouse model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3, treatment was initiated in which groups of 10 mice received vehicle (Group 1), CDDP at 6 mg kg (IV one time, Group 2), Compound 25 at 150 mg/kg in saline, once a week for two weeks (i.p., Group 3), or the combination of the two agents (Group 4).

[0474] The results shown in Figure 3 demonstrate that 150 mg/kg per week of Compound 25 provided greater reduction in tumor growth than CDDP alone and that the combination of the two agents resulted in added benefit. These results also indicate that during the two week period of dosing mean tumor volume did not change indicating complete inhibition of tumor growth. These data indicate that Compound 25 administered at 150 mg/kg day as monotherapy, once a week, is the most effective all the dosing regimens described in the preceding 'examples (Examples 47-52). Little change in body weight was observed suggesting reduced toxicity with this dosing regimen.

[0475] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective dose of about 12 mg/kg/day, for the treatment, of cancer, optionally administered at a frequency of once every week, particularly non-small cell lung cancer, alone or in combination with CDDP.

Example 54 : . 176

[0476] Example 54 describes efficacy of Compound 25 via an ip bolus injection or ip infusion alone or in combination with Cisplatin in H460 xenografts mouse model. Female Nu-Foxnlnu homozygous nu/nu mice (purchased from Charles River, Cambridge, MA), 6 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line HT29 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of mice (ten per group) was administered for three weeks, as tabulated in the table below: compound Compound 25 (formulated as a 15 mg/ml saline solution, administration route - i.p., administrated 2 h before CDDP on the days the combination therapy is scheduled and CDDP in saline, V.

Treatment protocol * - Alzet pump, 200 μϊ^ for 1 week x 2 (re-implant new pump at the end of one week).

[0477] ' The body weight and tumor volume was determined as described in Example 47. The results are indicated in Figure 4. The data indicate that, while continuous application of Compound 25 alone or in combination with CDDP is efficacious, intennitteiit, such as once a week dosing can provide greater therapeutic benefit in the treatment of certain cancers such as non-small cell lung cancer.

Example 55 Compound 25 And Gemcitabine Combination Therapy

[0478] A" combination of Compound 25 and gemcitabine was administered to nude mice that were carrying tumors derived from type MiPaca2 human pancreatic cancer cells.

MiaPaca-2 tumor is a highly invasive, rapidly growing tumor that results in death within 20- ί . : ■ 177 days in untreated animals. The tumor cells had been transfected with the gene for red fluorescent protein. Mice were administered doses of vehicle control, gemcitabine, Compound 25, Compound 24, or gemcitabine/ Compound 25 combinations or gemcitabine/ Compound 24 were administered i.p., as tabulated below (8 mice/ roup). Compounds 24 and 25 were formulated in saline and provided by Threshold Pharmaceuticals, Inc. as a dry powder. Gemcitabine was obtained commercially and prepared freshly according to manufacturer's instructions.

Treatment protocol * qd = every day; qw = every week.

[0479] Tumors were imaged once weekly until the end of the study at which time open body images were obtained to confirm effects. In Group 1, the tumors grew rapidly (Figure 5) and resulted in 100% lethality by day 30 (Figure 6).

[0480] Groups 3 and 4 resulted in minor effects on tumor volume and had little effect on survival. Group 2 significantly reduced tumor volume and prolonged survival. Group 6 provided modest reduction in tumor size but no additional effects on survival. In contrast, Group 5 demonstrated significantly reduced tumor growth and significantly prolonged survival compared to Group 2. Five out of S tumors in Group 5 regressed rapidly after i treatment and within a short period failed to emit fluorescence (Figure 7).

[0481] Four of these tumors remained at zero fluorescence until the end of the experiment and the tumors were considered to be cured. No tumors in Group 2 were considered to be cured. These results demonstrate that combination treatment with Compound 25 and gemcitabihe is of greater benefit in this model of cancer compared to monotherapy with the stand of care, gemcitabine. These results demonstrate that tumor reduction in animals administered with a combination of Compound 25 at 30 mg/mg/day and, gemcitabine is significantly greater than that in animals treated with gemcitabine as a single agent. 178

[0482] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective doses of about 2.5 mg/kg/day, for the treatment of cancer, particularly pancreatic cancer, in combination with gemcitabine.

Example 56

[0483] It is recognized that efficacious molecules for treatment of human diseases including cancer maybe toxic at doses near or sometime much greater than doses necessary to achieve beneficial effects. To determine appropriate dose and route of administering such a compound, it is necessary to understand its toxicity. Routinely, initial approaches to determining the toxic dose involve the use of rodents such as mice to provide preliminary data that might support the design of similar studies in larger animals and humans. Test compounds (Compounds 24, 25 and 36) were tested in mice as preliminary experiments for determining doses to be used in larger animals. Compound 25 was tested at doses as high as 300 mg/kg as a single dose and found to cause renal toxicities such as tubular necrosis and protein spillage into the urine. Transient reductions in white blood cells were also observed. However, little toxicity was noticed at lower doses (100 and 200 mg/kg). These doses selected represent an approximation of doses that might be used in larger animals such as rats and dogs for the purpose of confirming that such toxicities exist and for predicting if renal function should be measured in humans.

[0484] Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. All publications and patent documents (patents, published patent applications, and unpublished patent applications) cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission'that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims. 179

Claims (10)

1. CLAIMS:
2. 2. - 180-
3. 3. 188236/2
4. 4. A pharmaceutical formulation comprising the compound of claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent.
5. 5. The use of a compound of any one of clams 1 to 3 in the manufacture of a medicament for treatment cancer.
6. 6. A pharmaceutical formulation comprising the compound of claim 2 and a pharmaceutically acceptable excipient, carrier, or diluent.
7. 7. A pharmaceutical formulation comprising the compound of claim 3 and a pharmaceutically acceptable excipient, carrier, or diluent.
8. 8. The use of a compound of claim 2 in the manufacture of a medicament for treating cancer.
9. 9. The use of a compound of claim 3 in the manufacture of a medicament for treating cancer.
10. 10. A compound of any one of claims 1 to 3 for use in treating cancer. For the Applicants, REINHOLD COHN AND PARTNERS - 181 -